Advertisement
Journal of Clinical Oncology  
Search for:
Limit by:
  Browse by Subject or Issue
Home Search or Browse JCO My JCO Subscriptions Customer Service Site Map

Journal of Clinical Oncology, Vol 22, No 20 (October 15), 2004: pp. 4165-4173
© 2004 American Society of Clinical Oncology.
DOI: 10.1200/JCO.2004.01.035
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Save to my personal folders
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Osoegawa, A.
Right arrow Articles by Maehara, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Osoegawa, A.
Right arrow Articles by Maehara, Y.

Regulation of p27 by S-Phase Kinase-Associated Protein 2 Is Associated With Aggressiveness in Non–Small-Cell Lung Cancer

Atsushi Osoegawa, Ichiro Yoshino, Shinji Tanaka, Kenji Sugio, Toshifumi Kameyama, Masafumi Yamaguchi, Yoshihiko Maehara

From the Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka; and Department of Surgery II, Graduate School of Medical Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan

Address reprint requests to Ichiro Yoshino, MD, PhD, Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan; e-mail: iyoshino{at}surg2.med.kyushu-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
PURPOSE: The F-box protein S-phase kinase-associated protein 2 (Skp2) is one of the positive regulators of the cell cycle that promote ubiquitin-mediated proteolysis of the cyclin-dependent kinase inhibitor p27. In this study, we investigated the significance of Skp2 expression in human non–small-cell lung cancer (NSCLC).

PATIENTS AND METHODS: Clinicopathologic features and immunohistochemical expression of Skp2 and p27 proteins were studied in 138 patients with NSCLC. Survival analyses were performed using the Kaplan-Meier method and the Cox regression model. To analyze the role of Skp2 in vitro, NSCLC cells were transfected with an Skp2-expressing vector or small interfering RNA.

RESULTS: Skp2 was overexpressed in males, smokers, patients with squamous cell carcinomas, and patients with poorly differentiated cancers (P = .034, < .0001, < .0001, and .002, respectively). The multivariant analysis revealed that Skp2 expression is an independent prognostic factor for survival in NSCLC. An inverse relationship of Skp2 with p27 expression was observed (P = .012), and patients with both a higher expression of Skp2 and a lower expression of p27 showed a significantly unfavorable prognosis (P = .0002). In vitro ectopic expression of Skp2 in NSCLC cells reduced the protein level of p27. Conversely, induction of Skp2 siRNA increased the protein level of p27, leading to growth inhibition in NSCLC cells.

CONCLUSION: Skp2 overexpression is closely associated with the suppression of p27 and the aggressiveness in NSCLC. It also could be a therapeutic target in NSCLC.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
p27 protein is known to be a regulator of the eukaryotic cell cycle that acts by inhibiting cyclin and cyclin-dependent kinase complexes. Reduced expression of p27 protein is known as an independent prognostic marker in a large variety of cancers and is associated with unfavorable prognosis.1-5

Although levels of p27 protein oscillate during the cell cycle, p27 mRNA levels remain almost the same throughout the cell cycle. p27 protein levels are mainly regulated through degradation by ubiquitin-dependent proteolysis.6 A chain of ubiquitin adheres to the target protein by ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase. Then, the target protein is degradated by the 26-S proteasome.7 An F-box protein is the substrate recognition subunit of a ubiquitin ligase referred to as the S-phase kinase-associated protein (Skp) 1, cullin (Cul) 1, regulator of cullins (Roc) 1/RING box protein (Rbx) 1, and F-box protein (SCF) complex,6 and Skp2 has been identified as one of the F-box proteins required for the ubiquitination and consequent degradation of p27 both in vivo and in vitro.6

Several studies have indicated that there is a possible relationship between Skp2 expression and the aggressiveness of malignant tumors.8-17 Furthermore, both high levels of Skp2 and low levels of p27 have been related with a high grade of malignancy in oral squamous cell cancer, lymphoma, and gastric carcinoma.8-11

We previously reported that the reduced p27 protein level in tumor tissue was significantly associated with poor prognosis in curatively resected non–small-cell lung cancer (NSCLC).5 Here, we investigated Skp2 expression level, especially in regard to the relationship with the level of p27 expression in patients with NSCLC. In addition, we investigated whether Skp2 small interfering RNA (siRNA) could have a therapeutic use in NSCLC.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
Patients
Fresh surgical specimens from 138 consecutive Japanese patients with NSCLC were analyzed in this study. Informed consent was obtained from all patients (90 men and 48 women; age range, 37 to 82 years; mean age, 65.0 years). All patients had undergone treatment at the Department of Surgery and Science, Kyushu University Hospital (Fukuoka, Japan), from 1990 to 1993. The histology of the disease was determined based on hematoxylin and eosin–stained preparations according to the criteria of WHO.18 The result revealed that there were 78 adenocarcinomas, 55 squamous cell carcinomas, and five large-cell carcinomas. Pathologic staging was performed according to the tumor-node-metastasis classification system revised in 1997.19 It was determined that 62 patients had stage I disease, 29 had stage II disease, 38 had stage III disease, and eight had stage IV disease.

Immunohistochemistry
Immunohistochemical study was carried out on formalin-fixed, paraffin-embedded tissue sections. Four-micrometer sections were deparaffinized with xylene and rehydrated in a series of ethanols. Endogenous peroxidase was blocked in room temperature by 3% hydrogen peroxide in methanol for 20 minutes. Heat-induced epitope retrieval was performed in 0.01 mol/L citrate buffer (pH 6.0) for Skp2 immunostaining, and the samples were heated in a microwave oven at 99°C for 20 minutes. After blocking with normal rabbit serum, slides were incubated with a mouse monoclonal antibody against Skp2 (clone 1G12E9; Zymed Laboratories Ltd, San Francisco, CA) at the dilution of 1:100 at 4°C overnight. After washing the sections, they were treated for 30 minutes at room temperature in rabbit antimouse immunoglobulin. Staining for Skp2 was completed using the streptavidin-biotin-peroxidase complex method (Histofine SAB kit; Nichirei, Tokyo, Japan) with diaminobenzidine as a chromogene, and then the slides were counterstained with hematoxylin. For the negative controls, the primary antibody was replaced with phosphate-buffered saline containing 1% bovine serum albumin. A normal human tonsil was used as a positive control. At least 500 cancer cells in five high-power fields were counted, and the mean percentage of positively stained nuclei was 15.0% of all 138 samples (range, 0% to 79%). Hence, Skp2 protein expression was classified as positive when more than 15% of the cancer cell nuclei were stained and classified as negative when approximately 15% or fewer of the nuclei were stained. Immunohistochemical staining for p27 expression has been described previously.5 Briefly, after deparaffinization and rehydration, the sections were autoclaved at 121°C with 0.001 mol/L sodium citrate buffer (pH 6.0) for heat-induced epitope retrieval. Endogenous peroxidase was blocked by 1.5% hydrogen peroxidase in methanol for 10 minutes at room temperature. The primary antibody used was a mouse monoclonal antibody against p27 (NCL-p27, 1:100; Novocastra Laboratories Ltd, Newcastle upon Tyne, UK). The secondary antibody and the staining method were similar to those used for Skp2. The p27 protein expression was evaluated according to its intensity; expression was positive if the intensity of tumor cell nuclei staining was the same or stronger as that of infiltrating lymphocytes, and expression was negative if it was not.

Cell Culture and Transfection
A549, PC9, PC12 (human lung adenocarcinomas), and QG56 (human lung squamous cell carcinoma) cells were grown in RPMI 1640 medium (Sigma, Tokyo, Japan) containing 10% fatal bovine serum at 37°C in a 5% humidified CO2 atmosphere. HeLa (human cervical carcinoma) cells were grown in DMEM (Invitrogen, Carlsbad, CA), and those whole-cell lysates served as positive controls in Western blotting of Skp2 and p27.20

The human Skp2 expression vector pcDNA3.1-Skp2 (provided by Nakayama KI and Hatakeyamu S, Division of Cell Biology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan)21 was transfected into A549 cells by the Lipofectamine method (Life Technologies, Inc, Tokyo, Japan). Three stably transfected clones expressing abundant Skp2 protein were selected after 1 mg/mL of G418 (Life Technologies) treatment, and the clones were then used for subsequent experiments. Mock vector-transfected cells were used in bulk for the control.

Total RNA Isolation and Reverse Transcriptase Polymerase Chain Reaction (PCR)
Total RNA was extracted from each cultured cell homogenate in a state of subconfluency using the RNeasy mini kit (Qiagen, Valencia, CA), and the RNA was reverse-transcribed using murine leukemia virus reverse transcriptase. PCR amplification was performed using 25 cycles of incubation at 95°C for 1 minute, at 58°C for 1 minute, and at 72°C for 1.5 minutes. The quantity and quality of mRNA from all samples were certified by reverse transcriptase PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase gene. Amplification of p27 (215 bp) and Skp2 (341 bp) transcripts were carried out using the GeneAmp PCR system 9700 (Applied Biosystems, Foster, CA) and oligodeoxynucleotide primers (p27: forward, 5'-GGGGCTCCGGCTAACTCTG-3'; and reverse, 5'-GGCTTCTTGGGCGTCTG CTC-3'; and Skp2: forward, 5'-CCCTCCACGGCATACTGTC-3'; and reverse, 5'-ATTGGGGCATCTAACTAAA-3').

Protein Extraction and Western Blot Analysis
Logarithmically growing cultured cells were washed twice with ice-cold phosphate-buffered saline and then lysed with lysis buffer (0.5% Triton X-100; 50 mmol/L Tris, pH 7.6; 300 mmol/L NaCl; 2 mmol/L EDTA; and 2 mmol/L Na3VO4) containing phenylmethylsulfonyl fluoride (1 mmol/L), leupeptin (25 µg/mL), and aprotinin (25 µg/mL), and pepstatin A (3.5 µg/mL) for 25 minutes at 4°C. Cell lysates were clarified by centrifugation at 15,000 x g for 10 minutes at 4°C, and the protein content of the supernatants was determined by Bradford protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. Equal amounts of total protein (50 µg/lane) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the samples were transferred to polyvinylidene membranes (Immobilon; Millipore, Inc, Bedford, MA) at 2 mA/cm2 for 40 minutes at 4°C. After blocking the nonspecific binding sites by overnight incubation with Tris-buffered saline (25 mmol/L Tris and 150 mmol/L NaCl, pH7.5) containing 0.1% Tween-20 and 0.3% (for Skp2) or 5% (for p27 and beta-actin) nonfat milk, the membranes were blotted with mouse monoclonal antibodies against Skp2, p27 (clone 57; Transduction Lab, Lexington, KY), and beta-actin (clone AC-15; Sigma-Aldrich, St Louis, MO) for 1 hour at a dilution of 1:500, 1:2,500, and 1:5,000, respectively. Bound antibodies were detected with horseradish peroxidase–linked antimouse immunoglobulin (Amersham Pharmacia Biotech, London, UK). Signals were detected using ECL (Amersham Pharmacia Biotech) and analyzed with the use of LAS-3000 (Fujifilm, Tokyo, Japan), and the intensities of the blots were calculated by densitometry using Image Gauge software (Fujifilm).

RNA Interference
Logarithmically growing PC9 cells were seeded at a density of 1.0 x 105 cells/well in six-well plates in serum-containing medium. The siRNA used for Skp2 silencing was a 21-bp synthetic molecule corresponding to residues 756 to 776 of the Skp2 coding region. Skp2 siRNA was transfected 24 hours later in serum-free medium using Transmessenger transfection reagent (Qiagen) according to the manufacturer's instrument. After incubation for 3 hours at 37°C, the medium was changed to serum-containing medium. Cells were counted in triplicate wells after trypsinizing, and lysates were prepared, as described previously, at indicated times. A rhodamine-conjugated nonsilencing siRNA (Qiagen) was used for a negative control and for determining the transfection efficiency by fluoroscope. This experiment was repeated three times.

Statistical Analysis
Relationships among clinicopathologic factors and Skp2 and p27 expression were analyzed by the {chi}2 test and logistic regression analysis. Survival curves between subgroups divided by the expression level of Skp2 were drawn using the Kaplan-Meier method, and the significant difference among subgroups was compared using the log-rank test. The Cox proportional hazards multivariable regression model with the forward stepwise procedure was performed to select a set of independently significant prognostic factors. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
Protein Expression of Skp2 in NSCLC
The expression of Skp2 protein was immunohistochemically examined in 138 NSCLC patients (Fig 1). Skp2 expression was found only in neoplastic tissue specimens but not in normal tissue specimens. Positive expression of Skp2 was found in 57 patients (41%). Skp2 expression was significantly often detected in males, in patients with squamous cell carcinoma, and in smokers, compared with their counterparts, namely females, patients with adenocarcinoma, and nonsmokers (Table 1). The percentage of Skp2-positive results was significantly higher in males (48%) than in females (29%; P = .034). Patients with squamous cell carcinoma also had a higher Skp2-positive rate (65%) than patients with adenocarcinoma (22%; P < .0001). Smokers also showed a higher Skp2-positive rate (53%) than nonsmokers (13%; P < .0001). Well-differentiated carcinomas showed significantly infrequent expression of Skp2. There was no statistically significant relationship between pathologic stage and Skp2 expression.



View larger version (118K):
[in this window]
[in a new window]
  		Fig 1. Immunohistochemical staining for S-phase kinase-associated protein 2 (Skp2). Positive staining of nuclei of tumor cells for Skp2 was documented in a patient with adenocarcinoma and a patient with squamous cell carcinoma (upper lane). Negative staining of tumor cells was also documented (lower lane).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Relationship Among Clinicopathologic Factors, Skp2, and p27

 
Previously, we immunohistochemically demonstrated the expression of p27 protein in NSCLC patients5; here, we investigated the relationship between p27 expression and Skp2 expression using the same clinical samples. A significant negative relationship between Skp2 protein expression and p27 protein expression was observed (P = .012, Table 1). Skp2 was positive in 32 (53%) of 60 p27-negative patients. Conversely, Skp2 was negative in 53 (68%) of 78 p27-positive patients.

Previous studies have shown that Skp2 plays an important role in p27 proteolysis.22 Thus, we considered which factors might contribute to the reduced expression of p27 protein; the factors we analyzed in this study of 131 patients (five patients with large-cell carcinoma and two patients with data missing were excluded) were as follows: Skp2 expression, age, sex, histology, pathologic stage, differentiation, and smoking. Skp2-negative expression and adenocarcinoma were the only independent risk factors for the reduced expression of p27, with relative risks of 0.275 and 3.058, respectively (Table 2). Regarding Skp2 expression, a logistic regression revealed smoking and squamous cell cancers to be significant risk factors, with relative risks of 12.893 and 4.421, respectively (Table 3).


View this table:
[in this window]
[in a new window]
  		Table 2. Factor Analysis for the Expression of p27*

 

View this table:
[in this window]
[in a new window]
  		Table 3. Factor Analysis for the Expression of Skp2*

 
Prognostic Significance of Skp2 Protein Expression
A survival analysis was performed in 118 patients who underwent curative resection, in which 51 patients were Skp2 positive and 67 were Skp2 negative. The overall survival curves for the two groups are shown in Figure 2A. The 5-year survival rate in the Skp2-positive group was 37.8%, whereas it was 62.5% in the Skp2-negative group (P = .0009). The disease-free survival curves also revealed an unfavorable prognosis for the Skp2-positive group compared with the Skp2-negative group (5-year survival rates were 30.7% and 47.4%, respectively; P = .041). Next, the prognosis of subgroups divided according to the status of p27 and Skp2 expression was compared (Fig 2B). The Skp2-positive and p27-negative subgroup (n = 27) exhibited the lowest 5-year survival rate (29.5%), whereas the 5-year survival rate of the Skp2-negative and p27-positive subgroup (n = 46) was the highest (72.7%). Statistical significance was demonstrated in the Skp2-positive and p27-negative subgroup versus the Skp2-negative and p27-positive subgroup (P < .0001); statistical significance was also demonstrated in the Skp2-negative and p27-positive subgroup versus the Skp2-positive and p27-positive and Skp2-negative and p27-negative subgroups (P = .0041).



View larger version (16K):
[in this window]
[in a new window]
  		Fig 2. Postoperative survival of curatively resected non–small-cell lung cancers. Comparison was made according to the status of expression of (A) S-phase kinase-associated protein 2 (Skp2) and (B) p27/Skp2. *Wilcoxon test.

 
To compare the prognostic significance of variables such as Skp2 protein expression, age, sex, histology (adenocarcinoma v squamous cell carcinoma), pathologic stage (I v II to IV), differentiation (well and moderate v poor), and smoking (yes v no), a multivariate analysis was performed. As a result, Skp2 expression was shown to be a significant prognostic factor, with a relative risk of 2.035 (P = .0115), as well as pathologic stage (Table 4).


View this table:
[in this window]
[in a new window]
  		Table 4. Multivariate Analysis for Prognostic Factors*

 
Skp2-Dependent p27 Degradation in a Lung Cancer Cell Line
To analyze the role of Skp2 in p27 degradation in vitro, we generated a Skp2-transfected lung cancer cell line. A549, a lung adenocarcinoma cell line, had high p27 protein levels and low levels of Skp2 (Fig 3). Therefore, we transfected an Skp2-expressing plasmid vector to A549 cells using a lipofection method. Three stably transfected clones exhibited increased levels of both the protein and the mRNA of Skp2 compared with the mock transfectants. The amount of p27 protein was significantly reduced, although the expression of mRNA remained unchanged among the clones (Fig 4).



View larger version (33K):
[in this window]
[in a new window]
  		Fig 3. Western blot analysis of S-phase kinase-associated protein 2 (Skp2) and p27 in lung cancer cell lines. A549, PC9, and PC12 were human lung adenocarcinomas, and QG56 was human lung squamous cell carcinoma.

 


View larger version (27K):
[in this window]
[in a new window]
  		Fig 4. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis of S-phase kinase-associated protein 2 (Skp2) and p27 in three Skp2 transfectants and the control. Densitometric analyses for the Western blots (A) were shown in B (Skp2) and C (p27). Each protein level, except Skp2 protein level in clone 4 (marked by asterisk), varied significantly (P < .05) compared with the control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
RNA interference was carried out to investigate whether p27 protein levels would increase when PC9 and QG56 cells, which had high protein levels of Skp2 and low levels of p27 (Fig 3), were transfected with Skp2 siRNA. The siRNA transfection efficiency was as high as approximately 90% when the number of siRNA-transfected cells was counted through a fluorescence microscope at 4 hours after transfection (data not shown). Skp2 siRNA knocked down the protein levels of Skp2 in both PC9 and QG56 cells compared with control nonsilencing siRNA transfectants. It also increased the protein levels of p27 as we expected (Fig 5). These results were observed in all sample lysates, which were obtained on each day after transfection until the sixth day (data not shown).



View larger version (28K):
[in this window]
[in a new window]
  		Fig 5. Western blot analysis of QG56 and PC9 transfected with small interfering RNA (siRNA) of S-phase kinase-associated protein 2 (Skp2) or control. Each cell lysate was prepared at 3 days after transfection. Densitometric analyses for the Western blots (A) were shown in B (Skp2), and C (p27). Each protein level varied significantly (P < .05) compared with the control.

 
Growth Inhibition of Lung Cancer Cells by Skp2 RNA Interference
On the basis of the closed association between the Skp2 expression and the lung cancer progression in clinical samples, we finally examined the effects of RNA silencing of Skp2 on the growth of lung cancer PC9 cells. As demonstrated in Figure 6, the cell growth was affected in the Skp2 siRNA transfectants compared with nonsilencing siRNA transfectants.



View larger version (15K):
[in this window]
[in a new window]
  		Fig 6. Growth curve of PC9 cells. Cells were transfected with S-phase kinase-associated protein 2 (Skp2) small interfering RNA (siRNA) or control siRNA and counted in triplicate wells at indicated times after transfection. The representative data of three independent experiments are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
Authors' Disclosures of...
 REFERENCES
 
Here, we demonstrated that Skp2 protein was overexpressed in 41% of surgically resected specimens from NSCLC patients. It was found that males, smokers, patients with squamous cell carcinomas, and those with poorly differentiated cancers exhibited significantly higher levels of Skp2 protein. Previous studies have shown that Skp2 is overexpressed in several types of undifferentiated cancers.8 In NSCLC, it was reported that squamous cell carcinoma was associated with significantly higher Skp2 mRNA levels compared with normal lung tissue and adenocarcinoma.23 In this study, a logistic regression analysis revealed that both smoking and squamous cell carcinoma were the impact factors for higher expression of Skp2. Moreover, in patients with Skp2-positive adenocarcinomas, smoking was more prevalent than in patients with Skp2-negative adenocarcinomas (76.5% v 46.7%, respectively; P = .03). Taking these clinicopathologic data into consideration, it was speculated that Skp2 might be strongly affected by tobacco-derived materials. So far, it has been suggested that the expression of Skp2 protein is regulated at the transcriptional and posttranscriptional levels.24,25 Recently, GA-binding protein has been shown to bind Skp2 promoter element to enhance Skp2 promoter activity.26 The relationship between tobacco-derived carcinogens and the GA-binding protein activation should be elucidated because transcriptional activation of the GA-binding protein is supposed to be upregulated by p38 mitogen-activated protein kinase,27 and the mitogen-activated protein kinase has been shown to be activated by benzo(a)pyrene, one of the polycyclic aromatic hydrocarbons contained in cigarette smoke.28

Skp2 is the substrate-recognizing subunit of a ubiquitin ligase named SCF complex,6 and p27 is the target for ubiquitination and subsequent proteolysis by SCFSkp2.22 In this study, an inverse relationship between Skp2 and p27 protein was recognized not only in clinical cases, but also in our in vitro experiments using lung cancer cell lines. However, 25 (18%) of 138 patients showed elevated protein levels of both p27 and Skp2, and 28 (20%) of 138 patients showed decreased protein levels of both p27 and Skp2. This implied that other systems might also regulate p27 protein expression. Recent studies showed that cyclin-dependent kinase subunit (Cks)1 is also required for the ubiquitination of p27 by bridging Skp2 and its substrate, p27, in vivo and in vitro.29 Cks1-deficient mice have been shown to have reduced body weight and p27 accumulation,30 which have also been observed in Skp2-deficient mice.21 It was shown that levels of Cks1 mRNA were significantly high in patients with lung adenocarcinomas23; therefore, Cks1 may be much more closely related to p27 expression in adenocarcinomas. In addition, there are other factors than Cks1 included in p27 proteolysis. p27 is degraded by SCFSkp2 mainly during the G1/S transition in the nucleus; however, in the early part of the G1 stage, p27 is thought to be degraded in the cytoplasm in a Skp2-independent manner.31 This nuclear export of p27 might be regulated by jun-activating domain binding protein (Jab)1.32 Cks1, Jab1, and other factors, together with Skp2, might play important roles in p27 degradation. Further study is required to better understand the relationships between p27, Skp2, Cks1, and Jab1.

Like p27,1 Skp2 has been reported to be an independent prognostic factor in various types of malignancies such as gastric cancers, oral squamous cell cancers, and ovarian cancers.10,11,17 In our study, Skp2 protein expression is associated with poor prognosis in patients with NSCLC. Furthermore, the high Skp2 and low p27 group showed a more unfavorable prognosis, whereas the low Skp2 and high p27 group showed a better prognosis. A multivariate analysis revealed the significant finding that Skp2 was an independent prognostic factor in patients with NSCLC. Yet, there were other candidate targets for Skp2-mediated proteolysis. Skp2-deficient mice have been shown to have intracellular accumulation of p27 and cyclin E.21 Additionally, recent studies have revealed that Skp2 is responsible for the rapid turnover of Myc protein and its transcriptional activities.33,34 These substrates may enhance the aggressiveness of Skp2-overexpressed lung cancers and may also explain the ambiguous distribution of p27 and Skp2 expression in clinical samples.

RNA interference was first found in Caenorhabditis elegans, and recently, it has been applied for eukaryotic cell lines. A double-stranded and approximately 20 bp in length RNA, named siRNA, works for degradation of target mRNA and duplicates using RNA polymerase.35 We made an attempt to modulate Skp2 expression using the RNA interference procedure to consider its therapeutic use in the future. In the RNA interference experiment, p27 showed increased protein expression in Skp2 knocked-down lung cancer cells, QG56 and PC9. Furthermore, Skp2 siRNA–transfected PC9 cells exhibited an impaired growth capacity compared with controls. Taking together, these findings indicate that lung cancer aggressiveness was deteriorated by Skp2 RNA interference in vitro, possibly by inhibiting p27 proteolysis.

In conclusion, high expression of Skp2 protein is associated with reduced expression of p27 and the aggressiveness of disease in patients with NSCLC. Skp2 is an important candidate prognostic factor in patients with NSCLC, and it may provide a target for therapeutic agents such as Skp2 siRNA. Further studies are required to better understand the mechanisms involved in Skp2 expression and to determine the clinical applicability of Skp2 siRNA.


   Authors' Disclosures of Potential Conflicts of Interest
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
REFERENCES
 
The authors indicated no potential conflicts of interest.


   Acknowledgment
 
We thank Kei-Ichi Nakayama, PhD, and Shigetsugu Hatakeyama, PhD, for providing us with the Skp2-expressing vector.


   NOTES
 
Authors' disclosures of potential conflicts of interest are found at the end of this article.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
1. Ricardo VL, Lori AE, Long J, et al: P27Kip1: A multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am J Pathol 154: 313-323, 1999[Abstract/Free Full Text]

2. Slingerland J, Pagano M: Regulation of the cdk inhibitor p27 and its deregulation in cancer. J Cell Physiol 183: 10-17, 2000[CrossRef][Medline]

3. Catzavelos C, Tsao M, DeBoer G, et al: Reduced expression of the cell cycle inhibitor p27Kip1 in non-small cell lung carcinoma: A prognostic factor independent of Ras1. Cancer Res 59: 684-688, 1999[Abstract/Free Full Text]

4. Esposito V, Baldi A, De Luca A, et al: Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer. Cancer Res 57: 3381-3385, 1997[Abstract/Free Full Text]

5. Tsukamoto S, Sugio K, Sakada T, et al: Reduced expression of cell-cycle regulator p27Kip1 correlates with a shortened survival in non-small cell lung cancer. Lung Cancer 34: 83-90, 2001[CrossRef][Medline]

6. Pagano M, Tam S, Theodoras A, et al: Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269: 682-685, 1995[Abstract/Free Full Text]

7. Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem 67: 425-479, 1998[CrossRef][Medline]

8. Gstaiger M, Jordan R, Lim M, et al: Skp2 is oncogenic and overexpressed in human cancers. Proc Natl Acad Sci U S A 98: 5043-5048, 2001[Abstract/Free Full Text]

9. Latres E, Chiarle R, Schulman BA, et al: Role of the F-box protein Skp2 in lymphomagenesis. Proc Natl Acad Sci U S A 98: 2515-2520, 2001[Abstract/Free Full Text]

10. Kudo Y, Kitajima S, Sato S, et al: High expression of S-phase kinase-interacting protein 2, human F-box protein, correlates with poor prognosis in oral squamous cell carcinomas. Cancer Res 61: 7044-7047, 2001[Abstract/Free Full Text]

11. Masuda T, Inoue H, Sonoda H, et al: Clinical and biological significance of S-phase kinase-associated protein 2 (Skp2) gene expression in gastric carcinoma: Modulation of malignant phenotype by Skp2 overexpression, possibly via p27 proteolysis. Cancer Res 62: 3819-3825, 2002[Abstract/Free Full Text]

12. Hershko D, Bornstein G, Ben-Izhak O, et al: Inverse relation between levels of p27(Kip1) and of its ubiquitin ligase subunit Sko2 in colorectal carcinomas. Cancer 91: 1745-1751, 2001[CrossRef][Medline]

13. Chiarle R, Yan P, Piva R, et al: S-phase kinase-associated protein 2 expression in non-Hodgkin's lymphomas inversely correlates with p27 expression and defines cells in S-phase. Am J Pathol 160: 1457-1466, 2002[Abstract/Free Full Text]

14. Yang G, Ayala G, DeMarzo A, et al: Elevated Skp2 protein expression in human prostate cancer: Association with loss of the cyclin-dependent kinase inhibitor p27 and PTEN and with reduced recurrence-free survival. Clin Cancer Res 8: 3419-3426, 2002[Abstract/Free Full Text]

15. Sigenoretti S, DiMarcotullio L, Richardson A, et al: Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer. J Clin Invest 110: 633-641, 2002[CrossRef][Medline]

16. Yokoi S, Yasui K, Saito-Ohara F, et al: A novel target gene, SKP2, within the 5p13 amplicon that is frequently detected in small cell lung cancers. Am J Pathol 161: 207-216, 2002[Abstract/Free Full Text]

17. Shigemasa K, Gu L, O'Brien TJ, et al: Skp2 overexpression is a prognostic factor in patients with ovarian adenocarcinoma. Clin Cancer Res 9: 1756-1763, 2003[Abstract/Free Full Text]

18. World Health Organization: Histological Typing of Lung and Pleural Tumours (ed 3). Geneva, Switzerland, Springer-Verlag, 1999

19. Mountain CF: Revisions in the international system for staging lung cancer. Chest 111: 1710-1717, 1997[Abstract/Free Full Text]

20. Carrano AC, Eytan E, Hershko A, et al: SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1: 193-199, 1999[CrossRef][Medline]

21. Nakayama K, Nagahama H, Minamishima YA, et al: Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication. EMBO J 19: 2069-2081, 2000[CrossRef][Medline]

22. Bloom J, Pagano M: Deregulated degradation of the cdk inhibitor p27 and malignant transformation. Semin Cancer Biol 13: 41-47, 2003[CrossRef][Medline]

23. Inui N, Kitagawa K, Miwa S, et al: High expression of Cks1 in human non-small cell lung carcinomas. Biochem Biophys Res Commun 303: 978-984, 2003[CrossRef][Medline]

24. Zhang H, Kobayashi R, Galaktionov K, et al: P19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell 82: 915-925, 1995[CrossRef][Medline]

25. Wirbelauer C, Sutterluty H, Blondel M, et al: The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: Evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts. EMBO J 19: 5362-5375, 2000[CrossRef][Medline]

26. Imaki H, Nakayama K, Delehouzee S, et al: Cell cycle-dependent regulation of the Skp2 promoter by GA-binding protein. Cancer Res 63: 4607-4613, 2003[Abstract/Free Full Text]

27. Flory E, Hoffmeyer A, Smola U, et al: Raf-1 kinase targets GA-binding protein transcriptional regulation of the human immunodeficiency virus type 1 promoter. J Virol 70: 2260-2268, 1996[Abstract]

28. Patten Hitt E, DeLong MJ, Merrill AH Jr: Benzo(a)pyrene activates extracellular signal-related and p38 mitogen-activated protein kinases in HT29 colon adenocarcinoma cells: Involvement in NAD(P)H:quinone reductase activity and cell proliferation. Toxicol Appl Pharmacol 183: 160-167, 2002[CrossRef][Medline]

29. Harper JW: Protein destruction: Adapting roles for Cks proteins. Curr Biol 11: R431-R435, 2001[CrossRef][Medline]

30. Spruck C, Strohmaier H, Watson M, et al: A CDK-independent function of mammalian Cks1: Targeting of SCF(Skp2) to the CDK inhibitor p27Kip1. Mol Cell 7: 639-650, 2001[CrossRef][Medline]

31. Hara T, Kamura T, Nakayama K, et al: Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway. J Biol Chem 276: 48937-48943, 2001[Abstract/Free Full Text]

32. Tomoda K, Kubota Y, Kato J: Degradation of the cyclin-dependent-kinase inhibitor p27(Kip1) is instigated by Jab1. Nature 398: 160-165, 1999[CrossRef][Medline]

33. von der Lehr N, Johansson S, Wu S, et al: The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription. Mol Cell 11: 1189-1200, 2003[CrossRef][Medline]

34. Kim SY, Herbst A, Tworkowski KA, et al: Skp2 regulates Myc protein stability and activity. Mol Cell 11: 1177-1188, 2003[CrossRef][Medline]

35. Hannon GJ: RNA interference. Nature 418: 244-251, 2002[CrossRef][Medline]

Submitted January 8, 2004; accepted August 4, 2004.




This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
T. Fujita, W. Liu, H. Doihara, and Y. Wan
Regulation of Skp2-p27 Axis by the Cdh1/Anaphase-Promoting Complex Pathway in Colorectal Tumorigenesis
Am. J. Pathol., July 1, 2008; 173(1): 217 - 228.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
H.-C. Chang, F.-R. Chang, Y.-C. Wang, M.-R. Pan, W.-C. Hung, and Y.-C. Wu
A bioactive withanolide Tubocapsanolide A inhibits proliferation of human lung cancer cells via repressing Skp2 expression
Mol. Cancer Ther., May 1, 2007; 6(5): 1572 - 1578.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Thorac. Surg.Home page
I. Yoshino, D. Kawano, T. Oba, K. Yamazaki, T. Kometani, and Y. Maehara
Smoking Status as a Prognostic Factor in Patients with Stage I Pulmonary Adenocarcinoma
Ann. Thorac. Surg., April 1, 2006; 81(4): 1189 - 1193.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. Matsunobu, K. Tanaka, T. Nakamura, F. Nakatani, R. Sakimura, M. Hanada, X. Li, T. Okada, Y. Oda, M. Tsuneyoshi, et al.
The Possible Role of EWS-Fli1 in Evasion of Senescence in Ewing Family Tumors
Cancer Res., January 15, 2006; 66(2): 803 - 811.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Save to my personal folders
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Osoegawa, A.
Right arrow Articles by Maehara, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Osoegawa, A.
Right arrow Articles by Maehara, Y.

About
JCO		 Editorial
Roster		 Advertising
Information		 Librarians &
Institutions		 Rights &
Permissions		 PDA Services

Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
Terms and Conditions of Use
  HighWire Press HighWire Press™ assists in the publication of JCO Online