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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 Regulation of p27 by S-Phase Kinase-Associated Protein 2 Is Associated With Aggressiveness in Non–Small-Cell Lung CancerFrom 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
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.
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 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
Cell Culture and Transfection 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)
Protein Extraction and Western Blot Analysis
RNA Interference
Statistical Analysis
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.
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).
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).
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).
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).
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).
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.
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.
The authors indicated no potential conflicts of interest.
We thank Kei-Ichi Nakayama, PhD, and Shigetsugu Hatakeyama, PhD, for providing us with the Skp2-expressing vector.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
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. 
