Imaging, Diagnosis, Prognosis

Significance of Skp2 Expression in Primary Breast Cancer Hideto Sonoda, Hiroshi Inoue, Kazuhiko Ogawa,Tohru Utsunomiya Taka-aki Masuda, and Masaki Mori

Abstract Purpose: We previously reported the p27 expression level to be an independent prognostic factor, and a high S-phase -associated 2(Skp2)expression level was significantly correlated with a poor prognosis in patients with gastric cancer. We herein examined the Skp2expression in breast cancer and attempted to identify any associations between the Skp2 expression status and either the clinicopathologic variables or the patient’s prognosis. Experimental Design: We established four Skp2-transfected breast cancer cell lines and assessed the correlations between the Skp2and p27expressions using real-time reverse transcription-PCR and a Western blot analysis. We then analyzed the clinicopathologic significance of Skp2mRNA expression in 169 Japanese patients with breast cancer. An immunohistochemical analysis was also done. Results: The p27 protein expression markedly decreased after Skp2 transfection, whereas no alteration in the p27 mRNA expression was observed. The Skp2 protein expression level as determined by immunohistochemical staining thus showed a significant correlation with the Skp2mRNA expression ( P = 0.001) and a significant inverse correlation with the p27 protein expression (P = 0.042). The patients with a high Skp2 expression were significantly younger than those with a low expression (P = 0.002). The prognosis of patients with a high Skp2expression was significantly ( P = 0.022) poorer than for those with a low expression. Moreover, a high expression of Skp2was an independent variable that correlated with a shorter disease-free survival (relative risk, 3.33; 95% confidence interval,1.296-8.578; P =0.013). Conclusions: The present results suggest that Skp2may play an important role particularly in young breast cancer, and it is also considered to have strong independent prognostic potential and thus may prove to be a useful target for the treatment of breast cancer.

Breast cancer incidence rates vary substantially, with the One cyclin kinase–associated protein, p27, has been shown highest rates found in the United States, Canada, and Northern to have an inhibitory activity of cyclin/cyclin-dependent kinase Europe. In Japan, breast cancer recently surpassed stomach complex during the G0 and G1 phase (3). A number of studies, cancer to become the most popular female cancer in 1995 including our recent report (4), have shown the protein level (1, 2). In the past decade, breast-conserving treatment has of p27 to markedly decrease in a variety of cancers, such as become the standard treatment for early breast cancer, and breast, colon, prostate, lung, ovary, brain tumors, and the relapse rate has decreased thanks to advances in adjuvant lymphomas (5–16). In those reports, the protein level of p27 therapies, such as chemotherapy, hormonal therapy, and was down-regulated; however, the mRNA level is not always radiation therapy. Relapses, whereas treatable, are not curable suppressed accordingly. The reason of this discordance has by the currently available therapy. Differences have been been ascribed to the proteolysis system of p27. The protein suggested to exist in the biological properties of each type of level of p27 has recently been reported to mainly be regulated breast cancer. through the degradation by -dependent proteolysis (17–19), and S-phase kinase-associated protein 2 (Skp2) was identified as a cofactor that targets p27 for ubiquitination (20–22). The role of p27 in control has been

Authors’Affiliation: Department of Surgery, Kyushu University Hospital at Beppu, elucidated in a Skp2 knockout mice model (23). The cells from Beppu, Japan the knockout mice showed high levels of p27, free cyclin E, Received 8/5/05; revised 12/2/05; accepted 12/2/05. polyploidy, and centrosome overduplication, and these alter- Grant support: Scientific Research (B) grants-in-aid 15390398, 15390379, and ations were abolished in Skp2/p27 double knockout mice (24), 16390381;Scientific Research (C) grants-in-aid15591412and15591411;andJapan thus suggesting that p27 is a primary target of Skp2. Society for Promotion of Science’s grant-in-aid for exploratory research 16659337. The costs of publication of this article were defrayed in part by the payment of page A reduction in the p27 expression level is associated with a charges. This article must therefore be hereby marked advertisement in accordance high aggressiveness and a poor prognosis in various malignant with 18 U.S.C. Section 1734 solely to indicate this fact. tumors, including breast cancer (5, 25). Previously, we reported Requests for reprints: Masaki Mori, Department of Surgery, Kyushu University that the p27 expression status was an independent prognostic Hospital at Beppu, Beppu 874-0838, Japan. Phone: 81-977-27-1650; Fax: 81-977-27-1651; E-mail: [email protected]. factor for patients with gastric cancer (4). On the other hand, F 2006 American Association for Cancer Research. Esteva et al. reported that p27 expression was not related to the doi:10.1158/1078-0432.CCR-05-1709 disease-free survival (26). Furthermore, we showed that Skp2

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Fig. 1. RT-PCR (A) and western blot (B) of Skp2transfectants ( t) and mocks (m) of four breast cancer cell lines (SKBR3, CRL1500, YMB-1, MCF 7 ) and the relationship between Skp2and p27 expression in vitro.

mRNA was overexpressed in cancer tissue, and the higher Skp2 resistance to apoptosis, and invasion potential than the control expression group showed a significantly poorer prognosis in cells. These findings indicate that Skp2 expression can patients with gastric cancer, whereas gastric cancer cells trans- modulate the malignant phenotype of cancer, possibly via fected with Skp2 showed a significantly higher growth rate, p27 proteolysis (27). In addition, Signoretti et al. have recently

Fig. 2. Relationship between Skp2and p27 expression in breast cancer tissues. Immunohistochemical stainings for Skp2 (A, C, E, and G) and p27 (B, D, F, and H) of four cancers representative of three expression patterns of Skp2and p27.The cancers in the top row (A and B)show strong Skp2immunostaining and weak p27 immunostaining.The cancer in the second row (C and D)showsweak Skp2immunostaining and strong p27 immunostaining.The cancer in the third row (E and F) is weak for both Skp2and p27 immunostaining.The cancer in the fourth row (G and H) is strong for both Skp2and p27 immunostaining. Â100.

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iCycler iQ detection system (Bio-Rad, Richmond, CA) and iQ SYBR Ta b l e 1. Relationship among the expressions of Skp2 Green Supermix. The reactions were done in 96-well plates with Optical- protein, Skp2mRNA, and p27protein in breast Quality 8-tube Strips (Bio-Rad). Skp2 and glyceraldehyde-3-phosphate cancers (n =137) dehydrogenase mRNA were amplified in separate tubes using the following protocol: 95jC for 10 minutes, then 35 cycles of denaturation at 95jC for 30 seconds, annealing at 60jC for 30 Skp2 mRNA P seconds, and extension at 72jC for 30 seconds. The increase in >1. 0 2 V1. 0 2 fluorescence was measured in real-time during the extension step. The following primers were used (all 5V to 3V direction): Skp2 sense primer, Skp2protein GCTGCTAAAGGTCTCTGGTGT and antisense primer, AGGCTTA- Strong 47 25 <0.001 GATTCTGCAACTTG; glyceraldehyde-3-phosphate dehydrogenase sense Weak 8 57 primer, GTCAACGGATTTGGTCTGTATT and antisense primer, p27 protein AGTCTTCTGGGTGGCAGTGAT. Strong 15 46 0.042 Immunohistochemistry. From the 169 breast cancer cases that were Weak 38 38 analyzed for their Skp2 mRNA expression level, we selected 137 specimens of which formalin-fixed, paraffin-embedded tissue was available. Immunohistochemical studies of Skp2 and p27 were done using the avidin-biotin-peroxidase method (LSAB kit, DAKO, Kyoto, showed that Skp2 is overexpressed in breast cancers that are Japan) as described previously (4). All sections were counterstained negative for both the estrogen and HER-2 receptor and with with hematoxylin. The primary monoclonal antibodies against Skp2 either the primary and local recurrences or metastatic disease and p27 were used at dilutions of 1:500 and 1:1,000, respectively. Skp2 from breast cancers. A relationship between the Skp2 expres- and p27 scores were determined by observing 1,000 cancer cells in sion level and a poor prognosis was also observed (28). at least five high-power fields and were classified as high (staining in In the present study, we thus analyzed the Skp2 expression status in 169 Japanese patients with breast cancers and clarified any correlations with the clinicopathologic features, Ta b l e 2 . Skp2mRNA expression and including age, menopause, tumor size, the presence of lymph clinicopathologic factors in breast cancers node metastasis and estrogen and progesterone receptors, invasion to the vessels, disease-free survival, and overall Skp2 T value survival. A multivariate analysis using the Cox regression V P model was also done. 1. 0 2 >1. 0 2 (n =84) (n =85) Age (y) 58.2 F 1. 3 5 2. 8 F 1.1 0.0 0 2 Materials and Methods Size (cm) z2.1 48 47 NS (0.809) Clinical samples. Fresh surgical specimens were obtained from 169 <2.1 36 38 patients with primary breast cancer after informed consent was Menopause obtained. The patients had all undergone surgery at the Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Premenopausal 25 35 NS (0.121) Kyushu University (Beppu, Japan) from October 1993 to October 1999. Postmenopausal 59 50 The patients with advanced stage were treated with postoperative Lymph node metastasis hormonal therapy combined with radiotherapy. Chemotherapy com- Present 37 39 NS (0.811) bined with radiotherapy was done when they had recurrent disease. Absent 47 46 Data concerning the patient characteristics, including age, menopause, Lymphatic involvement tumor size, the presence of lymph node metastasis and estrogen and Present 40 34 NS (0.318) progesterone receptors, invasion to the vessels, disease-free survival, Absent 44 51 overall survival, and development of metastasis, were available for Vascular involvement all 169 patients, and the observation period ranged from 7.5 to 131 Present 1214 NS (0.694) months (the median follow-up period was 34.7 months). Of the 169 patients, 38 showed recurrence, and 20 died of breast cancer. Absent 7271 Cell culture. Human breast cancer cell lines YMB-1, MCF 7, SKBR3, Estrogen receptor and CRL1500 were obtained from the Cell Resource Center for Present 47 37 NS (0.267) Biomedical Research Institute of Development, Aging and Cancer Absent 20 25 (Tohoku University, Sendai, Japan). YMB-1was maintained in DMEM, No data 17 23 and MCF 7, SKBR3, and CRL1500 were maintained in RPMI 1640 Progesterone receptor supplemented with 10% fetal bovine serum at 37jCina5% Present 4237 NS (0.2 67) humidified CO2 atmosphere. Absent 25 25 Antibodies. Mouse monoclonal antibodies to Skp2 and p27 were No data 17 23 purchased from the Zymed Laboratory (San Francisco, CA) and Stage Transduction Laboratories (Lexington, KY), respectively. Total RNA isolation. Frozen tissue specimens or cultured cell lines I3133NS(0.205) in a state of subconfluency were homogenized, and the total RNA II 49 42 was extracted using the modified acid/guanidine/phenol/chloroform III 4 10 method as described previously (29). Quantitative real-time PCR. The reverse transcriptase reaction was Abbreviation: NS, not significant. done as described previously (29). Real-time PCR was done using the

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Fig. 3. Disease free survival of patients with breast cancer at Stage I to IIIaccording to Skp2mRNA expression. High Skp2 mRNA expression group; T value > 1.0 2( n = 85) Low Skp2 mRNA expression group; T value V1.0 2( n =84)P =0.022; Mantol-Cox method.

>50% of cells) or low (staining in <50% of cells) as described carried out using the Mantel-Cox test. A multivariate analysis using previously (4). the Cox regression model was done only on the variables with Western blot analysis. Total protein was extracted from samples with showing P < 0.05 in the univariate analyses. All statistical differences radioimmunoprecipitation assay buffer. Aliquots of total protein were were deemed significant at the level of P < 0.05. The staging of applied to 10% acrylamide gradient gels. After electrophoresis, samples breast cancers were classified based on the criteria established by the were electroblotted onto a polyvinylidene membrane (Immobilon, Union Internationale Contre le Cancer. Millipore, Inc., Bedford, MA) at 0.5 A for 40 minutes at 4jC. Skp2 and p27 were detected using the mouse monoclonal antibodies at a dilution of 1:2,000 and 1:2,500, respectively. The blots were developed with Results horseradish peroxidase–linked antimouse immunoglobulin (Promega, Inc., Madison. WI). The signals were detected using Supersignal (Pierce, Relationship between Skp2 and p27 expression in Skp2- Inc., Rockford, IL). transfected cells. We examined the Skp2 mRNA expression Transfection assays. Human Skp2 cDNA was generated by reverse in four breast cancer cell lines: YMB-1, MCF 7, SKBR3, and transcription-PCR and subcloned into pcDNA3.1expression vector CRL1500 with reverse transcription-PCR. All of these cell lines (Invitrogen, Carlsbad. CA) as described previously (27) and then were expressed a low level of Skp2 mRNA (data not shown). transfected transiently into the cell lines by the LipofectAMINE method (Life Technologies, Inc., Tokyo, Japan) as described previously (30). Following Skp2 transfection, the p27 protein expression A mock vector–transfected clone was used as a control. decreased in all four of the cell lines (Fig. 1), whereas there Statistical analysis. Associations between the variables were tested was no change in the p27 mRNA expression. by Student’s t test or Fisher’s exact test. Survival curves were drawn Skp2 and p27 expression in breast cancer. An immunohisto- according to the Kaplan-Meier method, and a survival analysis was chemical analysis in 137 breast cancer tissue specimens revealed

Fig. 4. Overall survival of patients with breast cancer at Stage I to III according to Skp2mRNA expression. High Skp2mRNA expression group;T value > 1.0 2( n = 85) Low Skp2mRNA expression group;T value V1.0 2( n =84) P = 0.130; Mantol-Cox method.

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Skp2 showed a decrease in the p27 protein levels with no Ta b l e 3 . Multivariate analysis on disease-free survival remarkable change in its mRNA expression levels. The Skp2 (n =169) protein levels correlated with the Skp2 mRNA expression and inversely correlated with the p27 protein levels in breast cancer Relative risk P tissue specimens. Furthermore, a high Skp2 mRNA expression (95% confidence interval) significantly correlated with a poor disease-free survival for stage Skp2 mRNA expression 3.334 (1.296-8.578) 0.0125 I to III primary breast cancer. It was previously reported that a Lymph node metastasis 3.994 (1.361-11.715) 0.0117 high Skp2 expression correlated with a poor disease-free survival Lymphatic involvement 1.723 (0.653-4.547) 0.2720 for laryngeal cancer (32), sarcomas (33), ovarian cancer (34), Vascular involvement 1.033 (0.398-2.681) 0.9464 and lymphoma (35) while showing a poor prognosis for oral Tu m o r s i z e ( >2.1cm) 2.021 (0.757-5.393) 0.1601 cancer (36), laryngeal cancer (32), sarcomas (33), ovarian cancer (34), lymphoma (35), lung cancer, gastric cancer (27), and breast cancer (28). Those reports analyzed at most 100 cases, whereas our present study thus outnumbered them. that Skp2 protein was predominantly expressed in breast cancer We consider it likely that Skp2 is related to a degradation of cells. The breast carcinoma cases were then classified into four p27, in vivo as well as in vitro, and it could thus be a potentially major patterns of Skp2/p27 expression (Fig. 2). Fifty-two tumors useful relapse marker for patients with breast cancer. A (38%) showed a strong Skp2 protein level and a weak p27 significant difference in age was found between the high Skp2 protein level. Thirty-eight tumors (28%) expressed a weak Skp2 mRNA expression group and the low expression group. Breast protein level and a strong p27 protein level. In 33 tumors (24%), cancer in young patients has been reported to be likely negative both weak Skp2 and weak p27 protein levels were observed. for estrogen receptor (37–41), whereas it also has a higher Fourteen tumors (10%) expressed both strong Skp2 and strong S-phase fraction than breast cancer in elderly patients (42, 43), p27 protein levels. The Skp2 protein level thus showed a strong and it also correlated with a poor prognosis (37, 40, 43). Our correlation with the Skp2 mRNA expression (P < 0.001), and it present data showed the Skp2 mRNA expression to be signi- was also significantly reverse correlated with the p27 protein ficantly higher in patients younger than 55 years (Table 2). expression level (P = 0.023). The p27 protein expression level Although no significant difference was observed, the high Skp2 showed the inverse correlation with the Skp2 mRNA expression mRNA expression group more frequently showed estrogen level (P = 0.042; Table 1). receptor–negative tumors than the estrogen receptor–positive Clinical significance of Skp2 expression in breast cancer. cases (P = 0.109). This finding is consistent with previous To perform a quantitative analysis, we evaluated the expression observation, as reported by Signoretti et al., on 84 patients with of Skp2 mRNA in tumor tissue specimens (T) by normalization advanced breast cancer (28), although they did not employ a as follows: T = Skp2 (T)/glyceraldehyde-3-phosphate dehydro- multivariate analysis. We thus did a multivariate analysis; thus, genase (T)/Skp2 of YMB-1/glyceraldehyde-3-phosphate dehy- high expression of Skp2 was found to be an independent drogenase of YMB-1as determined by a real-time PCR analysis. prognostic variable that correlated with a shorter disease-free According to this analysis, Ts ranged from 0 to 39 (median, survival (relative risk, 3.33; 95% confidence interval, 1.296- 1.02). We set a cutoff value (T, 1.02) for these mRNA expression 8.578; P = 0.013; Table 3). In addition, the high Skp2 mRNA levels in tumor tissue specimens, because the median level of expression group showed a poor disease-free survival. Skp2 mRNA in the 169 cancer tissue specimens was 1.02. Using We previously found that Skp2 transfection modulated the this cutoff value, we classified the tumors into two groups: high malignant phenotype of cancer cells. The increase in the expression group (n =85) and a low expression group (n = 84). percentage of cells in the , a high proliferation activity, As shown in Table 2, a significant difference in age was found resistance to apoptosis, and a high degree of invasiveness were between the high Skp2 mRNA expression group and the low specifically induced by Skp2 transfection in gastric cancer cells expression group (P = 0.002). On the other hand, no significant (27). The high Skp2 expression group was more frequently difference was seen regarding the tumor size, vascular or observed in the younger patients group. A high expression of lymphatic involvement, lymph node metastasis, or the state of Skp2 may thus play a more important role in breast cancers estrogen receptor or progesterone receptor. Of the 169 patients occurring in young patients. classified from stages I to III, the high Skp2 mRNA expression It is important to clarify the molecular mechanisms by which a group (n = 85) showed a significantly (P = 0.022) poorer Skp2 expression is up-regulated in breast cancer. Yokoi et al. disease-free survival than the low expression group (n = 84; showed that Skp2, located at 5p13, often showed genomic Fig. 3). In a multivariate analysis, the high Skp2 mRNA amplification not only in cell lines but also in primary small cell expression group showed a significantly poorer disease-free lung carcinomas (44). On the other hand, Pene at al. reported survival, independent of the existence of the lymph node that LY294002, a phosphorylation inhibitor of Akt, up-regulated metastasis at the time of operation (relative risk, 3.33; 95% the p27 protein expression, whereas the expression of both Skp2 confidence interval, 1.296-8.587; P = 0.013). However, no and cyclin D1dramatically diminished in vitro. This suggested significant correlation was observed between the overall that the biological effects of phosphatidylinositol 3-kinase survival and the expression Skp2 mRNA (Fig. 4). activation might participate in a reduction of Skp2 protein and an increase of p27 protein (45). Rosner et al. recently indicated Discussion that TSC2 (tuberous sclerosis complex 2) deletion can shorten p27 half-life because TSC2 can bind to p27 and interfere with Consistent with Krek’s and our previous transfection assays on p27 binding to Skp2 (46). Recent studies discovered a cyclin- cell lines (27, 31), the breast cancer cell lines transfected with dependent kinase subunit 1to be an integral part of the

www.aacrjournals.org 12 19 Clin Cancer Res 2006;12(4) February 15, 2006 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Imaging, Diagnosis, Prognosis ubiquitination mechanism for p27 (47, 48). In addition, the factor for breast cancer and also inversely correlated with the p27 relationship among Skp2, cyclin-dependent kinase subunit 1, expression in breast cancer. These findings strongly suggest that and p27 was also found to play a very important role in gastric Skp2 could play an important role in breast cancer progression and cancer carcinogenesis (49). may also be a novel molecular target for the treatment of breast In conclusion, the prognosis of patients with a high Skp2 cancer, especially in breast cancer occurring in young women. expression was significantly (P = 0.022) poorer than those with a low expression. Moreover, a multivariate analysis revealed that a Acknowledgments high expression of Skp2 was an independent variable that correlated with a shorter disease-free survival. Thus, a Skp2 gene We thank T. Shimooka, K. Ogata, J. Miyake, and M. Ikeda for their valuable overexpression could therefore be a poor disease-free survival technical assistance.

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