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High Expression of S-Phase Kinase-interacting Protein 2, Human F-box Protein, Correlates with Poor Prognosis in Oral Squamous Cell Carcinomas¹

Department of Oral Pathology, Hiroshima University, Faculty of Dentistry [Y. K., S. K., S. S., M. M., T. T.], and Clinical Laboratory, Hiroshima University Dental Hospital [I. O.], Hiroshima 734-8553, Japan

	ABSTRACT

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Reduced expression of p27^Kip1, a cyclin-dependent kinase (Cdk) inhibitor, is frequently found in various cancers, including oral squamous cell carcinoma (OSCC), and is attributable to an enhancement of its degradation. Skp2, an F-box protein necessary for DNA replication, is required for the ubiquitinylation and subsequent degradation of p27^Kip1. In the present study, we examined the expression of Skp2 and its correlation with the expression of p27^Kip1 protein or p27^Kip1 degradation in OSCC. Using immunohistochemistry, we found that high expression of Skp2 was present in 49% of OSCCs and only 20% of epithelial dysplasias. Significantly, high expression of Skp2 was correlated with poor prognosis of OSCC patients. We also found an inverse correlation between the expression of Skp2 and p27 by immunohistochemical analysis. A similar correlation was observed in OSCC cell lines and OSCC tissues by Western blot analysis. Interestingly, OSCC tissues with Skp2 expression had high p27^Kip1 degradation activity. These findings indicate that (a) Skp2 may play an important role for the development of OSCC, (b) Skp2 can be a novel target for OSCC treatment as well as a strong prognostic marker, and (c) the reduction in p27^Kip1 protein may be brought about by enhancement of its degradation mediated by increased levels of Skp2 protein.

	Introduction

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The proliferation and progression of cancer cells have been known to closely relate to abnormalities of various positive and negative cell cycle regulators. p27^Kip1, a Cdk³ inhibitor, mediates G₁ arrest induced by transforming growth factor ß, contact inhibition, or serum deprivation in epithelial cell lines (1 , 2) . The increase in the cellular abundance of p27^Kip1 upon induction of cell quiescence is primarily attributable to a decrease in the rate of its degradation (3) . p27^Kip1 is polyubiquitinated both in vivo and in vitro, and less p27^Kip1 ubiquitination activity is present in proliferating cells than in quiescent cells (3) . Furthermore, p27^Kip1 ubiquitination requires its phosphorylation on threonine-187 (4) . Reduced expression of p27^Kip1 has been found frequently in various cancers, and the lack of p27^Kip1 is suggested to be the result of an enhancement of its degradation (5) . Aggressive human cancers express low levels of p27^Kip1 because of its decreased stability (5) . We also found that reduced expression of p27^Kip1 was shown in 87% of OSCC cases and was correlated with its malignancy (6) . Importantly, reduced p27^Kip1 levels represent a powerful prognostic marker for poor survival in cancer patients.

Skp2, an F-box protein necessary for DNA replication, was originally identified as a protein that interacts with cyclin A (7) . Recently, it has been reported that p27^Kip1 is specifically recognized and targeted for ubiquitination by Skp2 (8, 9, 10) . Skp2 is required for the ubiquitination and subsequent degradation of p27^Kip1 both in vivo and in vitro (8, 9, 10) . Skp2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27^Kip1 (8) . Skp2 frequently is overexpressed in tumor cell lines, and forced expression of Skp2 in quiescent fibroblasts induces DNA synthesis (7 , 9) . These findings led us to hypothesize that the enhanced p27^Kip1 degradation observed in many aggressive human tumors might be attributable to increased levels of Skp2. However, the abnormal expression of Skp2 and the correlation between Skp2 and p27^Kip1 expression in cancer remain unclear. Therefore, in the present study, we examined the expression of Skp2 protein and its correlation with the expression of p27^Kip1 protein or p27^Kip1 degradation in OSCC.

	Materials and Methods

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Tissue Samples.
Tissue samples from 15 patients with epithelial dysplasias (12 men and 3 women) and 37 patients with OSCC (21 men and 16 women) were retrieved from the Surgical Pathology Registry of Hiroshima University Dental Hospital from 1976 to 1997. At the time of diagnosis, the ages of the patients with epithelial dysplasia and OSCC were 37–81 years (mean, 60.7 years) and 31–87 years (mean, 61.4 years), respectively. For the present analysis, only biopsied specimens from the tongue before radiochemotherapy were selected to avoid possible influences of the lesional sites and treatment modalities on data. Among the 37 OSCCs, follow-up data were available in 27 patients (15 men and 12 women). The mean follow-up period for these OSCC patients was 70.37 months (range, 4–209 months). For immunohistochemical examination, tissues were fixed in 10% buffered formalin and embedded in paraffin. The numbers of cases for each degree of epithelial dysplasia and histological grade of OSCC are listed in Table 1 . Histological grade and stage of tumor were classified according to the criteria of the Japan Society for Head and Neck Cancer (11) . Fresh samples were taken from neoplastic tissues and from nonneoplastic tissues for Western blot analysis.

Statistical Analysis.
Patients’ survival data were used to determine possible correlation between Skp2 or p27^Kip1 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 four groups with different Skp2 and p27^Kip1 expression were examined. The statistical significance of these data was measured by the Mantel-Cox test. Possible correlations between variables of the analyzed tumor samples were tested for association by the Fisher’s exact test. P < 0.05 was required for significance.

Preparation of Tissue Lysate.
In tissues from five OSCCs, we analyzed the expression of Skp2, its related p27 expression, and p27 degradation activity, using both cancerous tissues and corresponding nonneoplastic tissues. Frozen tissue samples were minced into small pieces, and then each sample was homogenized in cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 125 mM NaCl, 0.1% (v/v) NP40 (Sigma Chemical Co., St. Louis, MO), 5 mM EDTA, 0.1 M NaF, 10 µg/ml leupeptin (Sigma), 0.1 µg/ml trypsin inhibitor (Sigma), 0.1 µg/ml aprotinin (Sigma) and 50 µg/ml phenylmethylsulfonyl fluoride (Wako, Osaka, Japan). Lysates were incubated on ice for 1 h and spun down at 15,000 rpm for 5 min at 4°C. The protein concentration was determined by the Bradford protein assay (Bio-Rad, Richmond, CA) with BSA (Sigma) as a standard.

Cell Culture.
Six OSCC cell lines (HSC2, HSC3, HSC4, Ca9-22, Ho-1-U-1, Ho-1-N-1, and KB) were examined. All of the cell lines were provided by the Japanese Cancer Research Resources Bank. They were routinely maintained in RPMI 1640 (Kyokuto Pharmaceutical Industrial Co., Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Roche Diagnostics Australia Pty. Ltd., Castle Hill, Australia) and 100 units/ml penicillin-streptomycin (Life Technologies, Inc., Grand Land, NY), in a CO₂ incubator (5% CO₂ in air) at 37°C. For experiments, cells were grown to subconfluence in this medium. Cells were lysed as described above, and cell lysates were used for Western blot analysis.

Western Blot Analysis.
We examined the expression of Skp2 and p27^Kip1 protein in OSCC cell lines and tissue samples by Western blot analysis. Western blotting was carried out as described previously (13) . We used an antihuman Skp2 rabbit polyclonal antibody (diluted 1:100; Zymed Laboratories) and an antihuman p27^Kip1 mouse monoclonal antibody (diluted 1:100; Transduction Laboratories). Fifty µg of protein were subjected to 10% PAGE 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, United Kingdom) was used.

In Vitro p27^Kip1 Protein Degradation Assay.
The p27^Kip1 degradation assay was performed as described by Pagano et al. (3) . Each frozen tissue sample and cells were prepared by the addition of three to five volumes of lysis buffer containing 20 mM Tris-HCl (pH 8.5) and 1 mM DTT to a cell pellet. The following protease inhibitors were added: 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone; 10 µg/ml L-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone hydrochloride, and 1 µg/ml aprotinin. Each sample was frozen and thawed three times. The lysate was centrifuged at 13,000 rpm in an Eppendorf centrifuge for 15 min at 4°C. The supernatant was retrieved and stored at -80°C for the degradation assay. Purified histidine-tagged p27^Kip1 was incubated at 37°C for different times in 30 µl of a degradation mixture containing 100 µg of cell extract, 50 mM Tris-HCl (pH 8.5), 5 mM MgCl₂, 1 mM DTT, 2 mM ATP, 10 mM creatine phosphokinase (Sigma), 10 mM creatine phosphate (Sigma), and 5 µM ubiquitin (Sigma). p27^Kip1 degradation was analyzed by immunoblotting with an antihuman p27^Kip1 antibody.

	Results

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Immunohistochemical Expression of Skp2 in Epithelial Dysplasia and OSCC.
Fifteen cases of epithelial dysplasia of the tongue were analyzed, including 11 cases of mild to moderate, and 4 cases of severe dysplasia. Thirty-seven OSCC cases were also analyzed. The incidence of Skp2 expression in epithelial dysplasias and OSCCs is summarized in Table 1 . Of 15 epithelial dysplasia cases, 12 (80%) showed <30% positive cells (+ and -; Fig. 1A ). Only 1 (9%) of 11 mild-to-moderate dysplasia cases and 2 (50%) of 4 severe dysplasia cases showed high expression of Skp2 (>30% positive cells, ++; Fig. 1B ). We could not find loss of p27^Kip1 expression in the epithelial dysplasias.

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Immunostaining of Skp2 in epithelial dysplasias and OSCCs and correlation between Skp2 expression and survival rate. A, epithelial dysplasia (histology, mild to moderate; grading of Skp2 expression, +). Some epithelial cells in basal and granular layer show weak Skp2 expression. B, epithelial dysplasia (histology, severe; grading of Skp2 expression, ++). Most epithelial cells show intense Skp2 expression. C, OSCC (grading of Skp2 expression, ++). Most cancer cells show intense Skp2 expression. D, relationship of Skp2 to survival. Prognosis of patients with >30% Skp2 expression (n = 11; median survival, 11 months) was significantly poorer than prognosis for patients with <30% Skp2 expression (n = 17; median survival, 77 months). E, relationship of p27Kip1 to survival. Prognosis of patients with <5% p27Kip1 expression (n = 14; median survival, 34 months) was poorer than that for patients with >5% p27Kip1 expression (n = 13; median survival, 84 months). F, relationship of Skp2 and p27Kip1 expression to survival. Prognosis of patients with Skp2 >30%-p27Kip1 <5% (n = 6; median survival, 11 months) was poorer than the prognoses for other groups: Skp2 >30%-p27Kip1 >5% (n = 4; median survival, 10 months), Skp2 <30%-p27Kip1 <5% (n = 8; median survival, 64 months), and Skp2 <30%-p27Kip1 >5% (n = 9; median survival, 84 months). The statistical significance of these data was measured by the Mantel-Cox test.

Expression of Skp2 in OSCC Cell Lines and Tissues.
The expression of Skp2 and p27^Kip1 proteins in seven OSCC cell lines (HSC2, HSC3, HSC4, Ca9-22, Ho-1-N-1, Ho-1-U-1, and KB) was examined by Western blot analysis as shown in Fig. 2A . OSCC cells, except for HSC4 cells, showed expression of Skp2 protein. Cells lines with low-level p27^Kip1 expression (HSC2, HSC3, Ho-1-N-1, and KB) showed high expression of Skp2. Cells with high expression of p27^Kip1 (HSC4 and Ho-1-U-1) showed low expression of Skp2. We found an inverse correlation between Skp2 and p27^Kip1 expression in these cells. However, Ca9-22 cells showed high expression of Skp2 despite showing high expression of p27^Kip1.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Expression of Skp2 protein and correlation with p27Kip1 expression or p27Kip1 degradation activity in OSCC cell lines and tissues. A, expression of Skp2 and p27Kip1 protein in seven OSCC cell lines (HSC2, HSC3, HSC4, Ca9-22, Ho-1-N-1, Ho-1-U-1, and KB). B, expression of Skp2 and p27Kip1 protein in five OSCC tissues. C, p27Kip1 degradation activity in five OSCC tissues. Fifty µg of protein were subjected to Western blot analysis as described in "Materials and Methods." In vitro degradation assay of p27Kip1 was performed as described by Pagano et al. (4) . T, tumor tissue; N, corresponding nonneoplastic tissue.

	Discussion

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Skp2, a human F-box protein, was originally identified as a protein that interacts with cyclin A (7) . F-box proteins form an expanding family of eukaryotic proteins characterized by an 40-amino acid F-box motif that is necessary to bind the other subunits of the ubiquitin ligase (14) . F-box proteins are components of ubiquitin protein ligases (E3), called the SCF complex, which contains the following basic subunits: Skp1 (S-phase kinase-associated protein 1), a cullin subunit (called Cul1 in metazoans), Roc1 (also called Hrt1 or Rbx1), and one of many F-box proteins. Skp2 frequently is overexpressed in tumor cell lines, and forced expression of Skp2 in quiescent fibroblasts induces DNA synthesis (7 , 9) . In the present study, we demonstrated that high expression of Skp2 was found in 49% of OSCCs in comparison with 20% of epithelial dysplasias (Table 1 and Fig. 1 ). Moreover, high expression of Skp2 was well correlated with poor prognosis in OSCC patients (Fig. 1, D–F) . These finding are consistent with the following results indicating that Skp2 is an oncogene: (a) Skp2 was frequently overexpressed in tumor cell lines (7) ; (b) forced expression of Skp2 in quiescent fibroblasts induced DNA synthesis (9) ; (c) Skp2 cooperated with activated N-Ras in tumorigenesis in an in vivo model (15) ; (d) Skp2 cooperated with H-Ras^G12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice (16) ; and (e) cotransfection with Skp2 and cyclin E promoted abundant hepatocyte replication and hyperplasia of the liver in vivo (17) . There have been reports on overexpression of Skp2 protein in human tumors (7 , 16) ; however, our report is the first to show a correlation with prognosis. On the other hand, Skp2 may play an important role for regulating normal cell proliferation, because Skp2-deficient mice grow slower than littermate controls and have smaller organs, with all tissues containing decreased numbers of cell (18) . However, the mechanism of Skp2 overexpression in cancer cells is uncertain.

Recently, it has been reported that Skp2 is required for the ubiquitination and subsequent degradation of p27^Kip1 both in vivo and in vitro (8, 9, 10) . Skp2-/- cells show high levels of p27 and free cyclin E (non-Cdk2 bound), polyploidy, and centrosome overduplication (18) . It is well known that reduced expression of p27^Kip1 is frequently found in various cancers and correlated with poor survival of cancer patients (5) . Moreover, aggressive human cancers express low levels of p27^Kip1 because of its decreased stability (5) , but the detailed mechanism of abnormal degradation of p27^Kip1 protein in cancer cells is still unclear. We previously demonstrated that (a) reduced expression of p27^Kip1 was found frequently in OSCCs and was well correlated with poor prognosis (6) , (b) a reduction in p27^Kip1 protein was correlated with early invasion and metastasis of OSCC (12) , (c) in OSCC cell lines, reduced expression of p27^Kip1 protein was brought about by proteasome-mediated degradation (6) , and (d) p27^Kip1 accumulation by inhibition of proteasome function induced apoptosis in OSCC cell lines (13) . We hypothesize that reduced expression of p27^Kip1 protein observed in OSCC might be attributable to enhanced degradation mediated by increased levels of Skp2 protein. In this study, we found an inverse correlation between the expression of Skp2 and p27 by immunohistochemistry (Table 2) . Fifty-eight percent of cases without p27^Kip1 expression showed high Skp2 expression. We also found an inverse correlation in OSCC cell lines by Western blot analysis, but one of them (Ca9-22 cells) had high expression of Skp2 protein despite showing high p27^Kip1 expression (Fig. 2A) . In OSCC tissues, three of five cases showed an inverse correlation between the expression of Skp2 and p27 (Fig. 2B) . Interestingly, these three cases had high p27^Kip1 degradation activity (Fig. 2, B and C) . We think that the reduction in p27^Kip1 protein may be brought about by high Skp2-mediated degradation in some OSCC cases. It has recently been reported that human Cks1, a member of the Suc1/Cks family of proteins, which are essential for cell cycle progression, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2 (19) . We previously found overexpression of Cks1 mRNA in 15 (62.5%) of 24 gastric cancers (20) . Therefore, we think that another molecule such as Cks1 may be involved in p27^Kip1 degradation in OSCC cases who did not show high expression of Skp2 and showed reduced expression of p27^Kip1. To clarify the correlation between Skp2 expression and p27^Kip1 degradation, we plan to do further studies using more OSCC tissues.

In the present study, we found that high Skp2 expression was significantly correlated with poor prognosis in OSCC and an inverse correlation between the expression of Skp2 and p27^Kip1 in oral tissues. These findings indicate that (a) Skp2 may play an important role for the development of OSCC, (b) Skp2 can be a novel target for OSCC treatment as well as a strong prognostic marker, and (c) the reduction in p27^Kip1 protein may be brought about by enhancement of its degradation mediated by increased Skp2 protein.

	ACKNOWLEDGMENTS

 
We thank Dr. Pagano (New York University School of Medicine) for critically reading the manuscript and for reagents.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part of by Grants 12771085 and 13877317 from the Ministry of Education, Japan.

² To whom requests for reprints should be addressed, at Department of Oral Pathology, Hiroshima University Faculty of Dentistry, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Phone: 81 82 257 5631; Fax: 81 82 257 5619.

³ The abbreviations used are: Cdk, cyclin-dependent kinase; OSCC, oral squamous cell carcinoma; Skp2, S-phase kinase-interacting protein 2; Cks1, Cdk subunit 1.

Received 5/18/01. Accepted 8/13/01.

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