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© 2003 American Society for Clinical Oncology Skp2 Protein Expression in Soft Tissue Sarcomas
From the Department of Laboratory Medicine and Pathology and the Department of Oncology, Mayo Clinic, Rochester, MN. Address reprint requests to Ricardo V. Lloyd, MD, PhD, Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, 200 First St. SW, Rochester, MN 55905; email: lloyd.ricardo{at}mayo.edu.
Background: p45 S phase kinase-associated protein-2 (p45skp2), a member of the F-box family of proteins, is an important component of the Skp1-Cullin-F-box protein (SCF) ubiquitin-ligase complex (SCFskp2). The latter has been implicated in the ubiquitination and degradation of p27kip1 (p27) and G1-S cell cycle progression. The expression and prognostic role of Skp2 in a large series of soft tissue sarcomas has not been previously investigated. Methods: Clinicopathologic features and immunohistochemical expression of Skp2, p27, and Ki-67 proteins were studied in 182 cases of soft tissue sarcomas (American Joint Committee on Cancer stages II and III). Survival analyses were performed using the Kaplan-Meier method and the Cox regression model.
Results: The male to female ratio was 1.2:1, and the median age at the diagnosis was 53 years. The tumors were predominantly located in the lower extremities (n = 163; 90%) and had a median size of 9 cm. High Skp2 expression ( Conclusion: These results indicate that Skp2 expression is associated with cell proliferation and a worse prognosis in soft tissue sarcomas. The lack of an inverse correlation between Skp2 and p27 suggests that additional molecular events associated with either Skp2 expression or p27 proteolysis may be operating in these tumors.
A MEMBER OF the F-box family of proteins, p45 S phase kinase-associated protein-2 (p45skp2), is an important component of the Skp1-Cullin-F-box protein (SCF) ubiquitin-ligase complex (SCFskp2).1,2 The latter has been implicated in the ubiquitination and degradation of p27kip1 (p27), an important cyclin-dependent kinase (Cdk) inhibitor of the G1-S cell cycle transition.1–5 Recently, Skp2 was found to play an oncogenic role in the development of oral squamous cell carcinoma,6,7 colon cancer,8 and lymphoma.9 It has also been suggested that Skp2 tumorigenic properties are not restricted to p27 proteolysis.2,6,9 On the basis of these observations, we hypothesized that high expression of Skp2 would be associated with cell cycle progression and a poor prognosis in adult patients with soft tissue sarcomas.
One hundred and eighty-two patients diagnosed with nonvisceral soft tissue sarcomas (American Joint Committee on Cancer [AJCC]) stages II and III)10 treated at the Mayo Clinic were included in this series after institutional review board approval. Clinical data, original pathologic and surgical reports, and follow-up information were obtained for all patients. Hematoxylin-eosin–stained and immunostained archival slides were available for review in all cases for diagnostic confirmation. The tumors included myxoid-round cell, pleomorphic, and dedifferentiated liposarcomas (n = 61, 33%); leiomyosarcomas and myogenic sarcomas (n = 32, 18%); pleomorphic and undifferentiated sarcomas (n = 19, 10%); synovial sarcomas (n = 26, 14%); fibrosarcomas and myxofibrosarcomas (n = 23, 13%); and others (angiosarcoma, clear-cell sarcoma, extraskeletal osteosarcoma, epithelioid sarcoma, extraskeletal myxoid chondrosarcoma, and alveolar soft part sarcoma; n = 21, 12%).
Paraffin-embedded archival tissues were available from all tumors for immunohistochemical studies. Tissue sections were cut at 4 µm, treated with 0.1 mol/L citrate, pH 6.0, in a 800-W microwave oven for 15 minutes for antigen retrieval, and incubated with the primary antibodies overnight at room temperature. Antibodies used included monoclonal anti-Skp2 (Santa Cruz, Inc, Santa Cruz, CA; dilution 1:100), anti-p27 (Transduction Laboratory, Lexington, KY; dilution 1:1,000), and MIB-1 (Ki-67 antigen; AMAC, Westbrook, ME; dilution 1:50). Immunostaining was performed with the Elite avidin biotin peroxidase kit (Vector Laboratories, Inc, Burlingame, CA) according to manufacturer’s specifications. Positive controls included tonsil tissues stained for p27, Ki-67, and Skp2. Negative controls consisted of substituting normal serum for the primary antibody, which resulted in no staining of the tissues. Quantification of the percentage of cells expressing Skp2, p27, and Ki-67 was performed in an average of 2,500 cells/tumor sample and a two-scale grading system for the expression of these markers was established. The cutoffs for low and high level of expression were pre-established according to the median value of these markers in a preliminary series of 60 randomly selected tumors.11 Only moderate to intense nuclear staining for each marker was considered as a positive signal. High expression for Skp2, p27, and Ki-67 proteins was defined when
The statistical analyses were performed with the S-PLUS 6.0 software package (Insightful Corp, Reinach, Switzerland, 2001). Correlation and association analyses were performed using the Spearman rank correlation coefficient, Mann-Whitney U test, Kruskal-Wallis exact test, and Fisher’s exact test. Local recurrence-free survival, metastasis-free survival, disease-free survival (time until local recurrence or metastasis), and overall survival probabilities were calculated using the Kaplan-Meier product-limit method.12 The Cox univariate regression model was used to compare the above-described four survival end points between patients with tumors expressing high and low levels of Skp2, p27, Ki-67, and other clinicopathologic variables.12 Multivariate survival analyses were performed using the Cox multivariate regression model. A stepwise procedure was used to identify the minimum set of variables to be retained in the final regression model.12,13 Seventeen variables to be potentially included in the regression model were chosen before starting the study. They included Skp2, p27, Ki-67, patients’ age and sex, grade, tumor size and depth, AJCC stage, use of adjuvant chemotherapy and radiotherapy, type of surgery, and lines of cellular differentiation. The latter included lipoblastic (for well-differentiated/dedifferentiated, myxoid-round cell, and pleomorphic liposarcoma), myogenic (for leiomyosarcoma and myogenic sarcoma), fibroblastic (for fibrosarcoma and myxofibrosarcoma), synovial sarcoma, and pleomorphic-undifferentiated (for unclassified sarcomas and the so-called malignant fibrous histiocytoma). Histopathologic diagnoses were made on the basis of established criteria.14,15 Because soft tissue sarcomas comprise a heterogeneous group of mesenchymal tumors, the impact on survival of a specific tumor differentiation was evaluated independently of the histologic grade. For example, myxoid liposarcoma without round cell differentiation was coded as a low-grade tumor with lipoblastic differentiation, and pleomorphic liposarcoma was coded as a high-grade tumor with lipoblastic differentiation. All reported P values were two-sided, and statistical significance was set at P
Clinicopathologic Features The median age at diagnosis was 53 years (range, 1 to 88 years), and there were 100 males and 82 females (ratio, 1.2:1). The median tumor size was 9 cm (range, 1 to 33) and most tumors were located in the extremities (n = 163; 90%). One hundred and twelve tumors (62%) were considered high-grade neoplasms, and 70 tumors (38%) were considered low-grade neoplasms. The tumors were staged according to the AJCC staging system in stages II (101; 55%) and III (n = 81; 45%), and were excised with negative surgical margins according to the surgical pathology reports. Adjuvant radio- (median dose, 50 Gy) and anthracycline-based chemotherapy were administered in 82 patients (45%). The median follow-up was 6.2 years. Local recurrences or metastases developed in 119 patients (65%) after a median time of 3.0 years (5-year disease-free survival rate of 37%). Lungs were the most common site of metastases.
Skp2, p27, and Ki-67 Expression
Skp2 Expression and Survival By univariate analysis, high Skp2 expression (  10%) was associated with decreased metastasis-free, disease-free, and overall survival (Tables 3 , 5, and 7; Figures 2, 3, and 4). High Skp2 expression ( 10%) was also associated with decreased local recurrence-free survival (hazard ratio [HR] = 1.44), but statistical significance was not reached (Table 1). Other prognostic factors were analyzed for predicting local recurrence-free survival, metastasis-free survival, disease-free survival, and overall disease-specific survival by univariate analysis, and the results are shown in Tables 1, 3, 5, and 7, respectively.
In a multivariate model, Skp2 was an independent prognostic factor for local recurrence-free, disease-free, and overall survival (Tables 5  , 6, and 7). High Skp2 expression was also associated with metastasis-free survival (HR = 1.36), but statistical significance was not reached (Table 8).
Progression through the various phases of the cell cycle is controlled by phase transition surveillance mechanisms known as cell cycle checkpoints.4,16 At these points, DNA structural integrity is guaranteed before a new cell cycle phase is started.4,16,17 The most important checkpoint, which is commonly disrupted in human cancers, occurs at the G1-S transition where the retinoblastoma (Rb) tumor suppressor protein is sequentially hyperphosphorylated and inactivated by cyclin D-Cdk4/-Cdk6 and cyclin E-Cdk2 complexes.18,19 These holoenzymes are inhibited by a group of Cdk inhibitors of the Cip/Kip family, and p27 is an important member of this group.3,5,20–22 Despite constant intracellular levels of p27 mRNA throughout the entire cell cycle, p27 protein concentration undergoes marked changes, and is rapidly degraded late in the G1 phase.23 An important cellular event for p27 proteolysis and posterior ubiquitination is the phosphorylation of the Thr-187 at the carboxy-terminal portion of p27 by the cyclin E-Cdk2 complex.1,24–27 This posttranslational modification is required for p27 interaction with Skp2 protein, the F-box protein component of the SCFskp2 ubiquitin-ligase complex. F-box proteins seem to confer substrate specificity to the SCF complex; the presence of Skp2 is necessary for cyclin E and p27 ubiquitination and degradation.1,2,23 p27 expression has been shown to be a prognostic factor in a variety of neoplasms.28 Recently, we showed that p27 expression correlated with survival in myxoid-round cell liposarcomas.29 Kawauchi et al30 found similar results in a series of synovial sarcomas; the results have not been confirmed by others.31 Because p27 expression seems to be mainly regulated by posttranslational modification, and because of the recent investigations reporting the important role of Skp2 in this process,1,6–9,24–27 we investigated the expression of Skp2 in different tumors of mesenchymal differentiation. Skp2 expression correlated with both histologic grade and cell cycle progression measured by Ki-67 expression. No differential expression of Skp2 was found among the different histologic types after adjusting for histologic grade. In contrast to some other preliminary reports,8,9 we found no inverse correlation between Skp2 and p27 in our series. Similarly, we did not find an inverse correlation between p27 and Skp2 in a series of extra-abdominal soft tissue leiomyosarcomas in a separate study. In the same series, high expression of Skp2 correlated with cell proliferation and disease progression.32 These observations may be explained, at least in part, by additional oncogenic properties of Skp22,6,9,33 or by the presence of additional molecular events involving p27 proteolysis.34,35 Two groups36–38 have recently shown that the Cdk-binding protein Cks1 is necessary for p27 proteolysis by the SCFskp2 complex, and Hara et al34 described a Skp2-independent pathway for p27 proteolysis at the G0-G1 cell cycle transition. More complex mechanisms also seem to regulate p27 proteolysis. Malek et al35 recently suggested the existence of two sequential p27 proteolytic pathways with the use of mouse knock-in experiments: one pathway, stimulated by mitogens, operates from early to mid G1 independently of the p27 Thr-187 phosphorylation status; the other operates during late G1, S, and G2 in a Thr-187–dependent and mitogen-independent fashion. In addition, it has also been shown that p27 can be degraded by a ubiquitin-independent processing,39 and that its proteolysis can also occur in the nucleus.40 High Skp2 expression was associated with decreased local recurrence-free, metastasis-free, disease-free, and overall survival by univariate analysis, but statistical significance was not reached for local recurrence-free survival. In a multivariate model, high Skp2 expression became an independent prognostic factor for local recurrence-free survival and remained an independent predictor for both disease-free and overall survival after adjusting for several clinicopathologic variables, including specific cell differentiation, expression of the cell cycle–related markers Ki-67 and p27, and therapeutic procedures. Notably, for those outcomes in which Skp2 remained an independent prognostic factor, histologic grade was not significantly associated with an adverse outcome. The opposite was seen with regard to metastasis-free survival, for which high histologic grade remained an independent predictor of an adverse outcome. These observations, added to the fact that high Skp2 expression was more frequently observed in high-grade than in low-grade tumors, suggest an important role of Skp2 in more aggressive or poorly differentiated neoplasms. The more aggressive behavior of synovial sarcoma in comparison with other histologic types might be related to additional oncogenic properties of this tumor that are not apparently associated with Skp2 expression or cell proliferation. Skp2 is emerging as an important regulator of p27 expression and therefore of the G1-S cell cycle transition. Skp2 oncogenic properties have been demonstrated in initial studies of human cell cancers of different lineages, and its expression was associated with cell proliferation and a worse prognosis in this current series of mesenchymal neoplasms. The lack of an inverse correlation between Skp2 and p27 suggests that additional cellular events associated with Skp2 expression or p27 proteolysis may be operating in these tumors. New chemotherapeutic agents targeting Skp2 and other proteins involved in the regulation of the G1-S checkpoint41 might prove to be effective in the treatment of soft tissue sarcomas and other human cancers.
A recent study by Penn et al42 showed that overexpression of Skp2 in Karposi’s sarcoma was associated with stage but was not inversely correlated with P27 expression.
We thank Christine M. Lohse, Department of Biostatistics, Mayo Clinic and Mayo Foundation, for her assistance.
Andre M. Oliveira is currently at the Institute of Medical Genetics, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. This work was presented at the Sarcoma Scientific Session of the 2002 Annual Meeting of the American Society of Clinical Oncology.
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34. Hara T, Kamura T, Nakayama K, et al: Degradation of p27kip1 at the G0-G1 transition mediated by a Skp2-independent ubiquitination pathway. J Biol Chem 276:48937–48943, 2001 35. Malek N, Sundberg H, McGrew S, et al: A mouse knock-in model exposes sequential proteolytic pathways that regulate p27kip1 in G1 and S phase. Nature 413:323–327, 2001[CrossRef][Medline] 36. Ganoth D, Bornstein G, Ko T, et al: The cell-cycle regulatory protein Csk1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nat Cell Biol 3:321–324, 2001[CrossRef][Medline] 37. 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] 38. Bartek J, Lukas J: p27 destruction: Cks1 pulls the trigger. Nat Cell Biol 3:E95–E98, 2001[Medline]
39. Shirane M, Harumiya Y, Ishida N, et al: Down-regulation of p27kip1 by two mechanisms, ubiquitin-mediated degradation and proteolytic processing. J Biol Chem 274:13886–13893, 1999 40. Rodier G, Montagnoli A, Marcotullio L, et al: p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis. EMBO J 20:6672–6682, 2001[CrossRef][Medline] 41. Owa T, Yoshino H, Yoshimatsu K, et al: Cell cycle regulation in the G1 phase: A promising target for the development of new chemotherapeutic anticancer agents. Curr Med Chem 8:1487–1503, 2001[Medline] 42. Penn RM, Fernandez-Figueras MT, Puig MT, et al: Over-expression of P45 (Skp2) in Karposi’s Sarcoma correlates with higher tumor stage and extra cutaneous involvement but is not directly related to p27 (KIP1) down-regulation. Mod Pathol 15:1227–1235, 2002[CrossRef][Medline] Submitted May 16, 2002; accepted October 20, 2002. This article has been cited by other articles:
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
.05. 
