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Expression of survivin and Its Relationship to Loss of Apoptosis in Breast Carcinomas

Department of General and Gastroenterological Surgery, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan

	ABSTRACT

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Aberrant inhibition of programmed cell death (apoptosis) prevents normal homeostasis and promotes tissue tumorigenesis, but whether it also influences the outcome of common cancers has remained arguable. The expression of a novel IAP apoptosis inhibitor, survivin, in breast cancer and its association with tumor cell apoptosis and overall prognosis were examined in this study. Immunohistochemical analysis showed that survivin expression was positive in 118 of 167 cases (70.7%) of breast carcinomas of histological stages I to III. In contrast, no expression of survivin in adjacent normal tissue was detected. Although survivin expression was not correlated with p53 mutations, survivin-positive cases were strongly associated with bcl-2 expression (78.0% versus 47.5%; P = 0.0005) and reduced apoptotic index (0.62% ± 0.51% versus 1.27% ± 1.37%; P < 0.0001). In addition, patients with low apoptotic index (<0.52%) had worse survival rates than the group with high apoptotic index (0.52%; P = 0.028), and multivariate Cox proportional hazard model analysis identified apoptotic index as an independent prognostic factor (P = 0.024). The results suggest that apoptosis inhibition by survivin, alone or in cooperation with bcl-2, is a significant prognostic parameter of worse outcome in breast carcinoma.

	INTRODUCTION

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Abnormalities in the control of programmed cell death (apoptosis) play an important role in tumorigenesis (1 , 2) . This process involves an evolutionarily preserved multistep cascade and is regulated by proteins that promote or counteract apoptotic cell death (3) . bcl-2 was the first protein shown to lead to prolonged survival of cells by preventing apoptosis (4) . Several apoptosis inhibitors related to the baculovirus iap gene have been identified in humans, mice, and Drosophila (5 , 6) . Highly evolutionarily conserved, IAP² proteins contain two to three Cys/His BIRs and a COOH-terminal RING finger (4) . Recombinant expression of IAP proteins counteracted various forms of apoptosis in vivo (7) and in vitro (6) . These molecules are thought to block an evolutionarily conserved step in apoptosis. At least in the case of XIAP, this may involve direct inhibition of the terminal effectors caspase-3 and caspase-7 through a BIR-dependent recognition (8) . Recently, a novel gene encoding a structurally unique IAP apoptosis inhibitor, designated as survivin, has been identified. Survivin is an M_r 16,500 cytoplasmic protein with a single BIR and no RING finger. Recombinant expression of survivin prevents apoptosis induced by growth factor interleukin 3 withdrawal in a pre-B cell line (9) . Unlike bcl-2 (10) or other IAP proteins (5 , 6 , 7) , survivin is undetectable in terminally differentiated adult tissues but becomes notably expressed in the most common human cancers, including stomach (11) , colorectal (12) , lung, breast, pancreatic, and prostate cancers and high-grade non-Hodgkin’s lymphomas in vivo (9 , 13) . Our previous studies demonstrated that the expression of survivin was significantly associated with bcl-2 expression and reduced apoptotic indices, which were strongly correlated with poor prognosis after surgery in both gastric and colorectal cancers (11 , 12) . In this study, we assessed the expression of survivin in breast cancer and its potential effect on tumor cell apoptosis and overall survival.

	MATERIALS AND METHODS

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Patients and Samples.
We studied a series of 167 patients with invasive breast carcinomas who did not receive any form of treatment prior to surgery. The surgically resected specimens used for this study were obtained from consecutive patients with breast carcinoma who underwent potentially curative resection at the Department of General and Gastroenterological Surgery, Osaka Medical College Hospital, during the period from 1988 to 1994. Clinicopathological factors, tumor histologies, and disease stage were assigned according to the General Rules for Clinical and Pathological Recording on Breast Cancer (14) . The specimens consisted of 32 cases of stage I, 104 cases of stage II, and 31 cases of stage III tumors. All of the patients were female, and the mean age of the patients was 53.1 years (SD, 11.84 years; range, 29–89 years). With regard to postoperative adjuvant therapy, the patients with four or more axillary lymph node metastases had six cycles of CMF (100 mg/m² cyclophosphamide p.o. on days 1–14, 40 mg/m² i.v. methotrexate on days 1 and 8, and 500 mg/m² i.v. 5-fluorouracil on days 1 and 8, every 4 weeks) or CAF (100 mg/m² cyclophosphamide p.o. on days 1–14, 30 mg/m² Adriamycin i.v. on days 1 and 8, and 500 mg/m² 5-fluorouracil i.v. on days 1 and 8, every 4 weeks). The patients with three or less axillary lymph node metastases were treated with tegaful [(2-tetra-hydrofuryl)-5-fluorouracil] at an oral dose of 600 mg/day for 2 years after surgery. Node-negative premenopausal patients were followed without chemotherapy. Patients with ER-positive tumors and postmenopausal patients received adjuvant endocrine treatment with 20 mg/day oral dose of Tamoxifen. The median follow-up time in this study group was 96 months (range, 11–104 months). Routinely processed formalin-fixed, paraffin-embedded blocks containing the main tumor were prepared. Serial sections of 2–4 µm were cut from the blocks at the maximum cross-section of the tumor.

Immunohistochemical Staining for survivin and the Scoring Method for Its Expression.
A pilot study using the anti-survivin antibody was conducted on various neoplasms, including high-grade non-Hodgkin’s lymphoma, pancreatic cancer, colorectal cancer, and gastric cancer to confirm the specificity in staining tumor cells and to determine an appropriate dilution for mAb 8E2 (IgG1 kindly provided by Dr. D. C. Altieri, Department of Pathology, Yale University, School of Medicine, New Haven, CT) for staining of breast carcinomas. One case of stage III gastric cancer was stained intensively and reproducibly for survivin expression in >30% of tumor cells, and this was used as a positive control throughout the present study. Negative control slides processed without primary antibody were included for each staining. For immunohistochemical detection of survivin protein, the standard avidin-biotin-peroxidase complex technique was carried out by using an LSAB kit (Dako A/S, Carpinteria, CA). Before using the LSAB kit, antigen retrieval was done by the pressure cooking method as described previously (11 , 12) . In brief, deparaffinized and rehydrated sections were bathed in a 10^-3 M sodium citrate buffer (pH 6.0) after bringing the solution to a boil in a pressure cooker and boiled for 20 min while maintaining the pressure. After quenching in 3% hydrogen peroxide and blocking for 5 min, the sections were incubated overnight at 4°C with a 1:5 dilution of primary monoclonal antibody 8E2 raised against purified recombinant survivin. Biotinylated antimouse immunoglobulin and streptavidin conjugated to horseradish peroxidase were then added. Finally, 3,3'-diaminobenzidine was used for color development, and hematoxylin was used for counterstaining. Negative control slides processed without primary antibody were included for each staining. The scoring method for survivin was modified from that for bcl-2 expression described by Sinicrope et al. (15) . The mean percentage of positive tumor cells was determined in at least five areas at 400-fold magnification and assigned one of the following five categories: 0, <5%; 1, 5–25%; 2, 25–50%; 3, 50–75%; and 4, >75%. The intensity of survivin immunostaining was scored as follows: 1+, weak; 2+, moderate; and 3+, intense. Because tumors showed heterogeneous staining, the dominant pattern was used for scoring. The staining intensity of infiltrated lymphocytes for bcl-2 was judged as 3+ relative to the intensity of survivin staining. The scores indicating percentage of positive tumor cells and staining intensity were multiplied to produce a weighted score for each case. Cases with weighted scores <1 were defined as negative, and cases were otherwise defined as positive.

Immunohistochemical Staining for bcl-2 and p53.
For detection of bcl-2 and p53 protein, deparaffinized and rehydrated sections were immunostained using the same techniques as for survivin antigen staining. Monoclonal mouse antibody against bcl-2 (clone 124, diluted at 1:20; Dako, Copenhagen, Denmark) and a mouse antihuman p53 antibody (DO7, diluted 1:50; DAKO, Copenhagen, Denmark) were used as primary antibodies for bcl-2 and p53 immunostaining. Before addition of the primary antibodies, sections were heated in a microwave oven three times at 900 W for a total of 15 min in 10^-3 M sodium citrate buffer (pH 6.0). The other staining procedures were the same as those for survivin. The scoring criteria for bcl-2 were the same as those for survivin, and cases with weighted scores of <1 were judged as negative. For p53 expression, cases with <5% positively stained tumor cells were defined as negative; otherwise, they were defined as positive.

Histochemical Detection of Apoptosis and Determination of the AI.
Apoptotic cells and apoptotic bodies were detected by in situ labeling using an ApopTag in situ Detection kit (S7101-KIT; Oncor, Gaithersburg, MD). In brief, deparaffinized and rehydrated sections were digested with proteinase K (20 µg/ml in PBS; Wako, Osaka, Japan) for 20 min at room temperature and washed. After quenching in 3% hydrogen peroxide for 5 min, washing with PBS, and adding the equilibration buffer for 10 min, terminal deoxynucleotidyl transferase enzyme was pipetted onto the sections, which were then incubated at 37°C for 1 h. After stopping the reaction by putting sections in stop/wash buffer and washing, anti-digoxigenin-peroxidase was added to the slides. Finally, slides were washed with PBS, stained with diaminobenzine (DAKO A/S, Glostrup, Denmark) substrate, and counterstained with methyl green. A positive control was prepared by nicking DNA with DNase I (0.7 µg/ml; Stratagene Co., La Jolla, CA) for the first staining procedure. A specimen known to be positive for apoptotic cells was used as positive control for subsequent staining. Substitution of terminal deoxynucleotidyl transferase with distilled water was used as negative control. The AI was expressed as the ratio of positively stained tumor cells and bodies to all tumor cells, given as a percentage for each case, and determined according to the criteria described previously (11 , 12 , 16) . In brief, a minimum of 3000 cells was counted at 400-fold magnification. Positively staining tumor cells with the morphological characteristics of apoptosis were identified using standard criteria, including chromatin condensation, nuclear disintegration, and formation of crescent caps of condensed chromatin at the nuclear periphery.

Statistical Analysis.
All statistical analysis was performed using the SPSS 6.1 J software package for Macintosh (SPSS, Inc., Chicago, IL). Variables associated with survivin expression as well as the correlation between survivin and p53 or bcl-2 expression were analyzed by the ² test. Differences in tumor cell AI for groups differing according to survivin expression were checked by the independent Wilcoxon method. The survival curves were plotted according to Kaplan-Meier method and checked by the log-rank test. A value of P < 0.05 was considered statistically significant.

	RESULTS

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State of Expression of Survivin in Breast Carcinomas.
Immunohistochemical staining revealed that anti-survivin mAb 8E2 specifically reacted with breast carcinoma cells, with positive staining of the cytoplasm of cancer cells (Fig. 1a) , whereas no expression of survivin was observed in adjacent normal tissues. Unlike bcl-2, no reactivity of the anti-survivin mAb 8E2 with infiltrating lymphocytes and other stroma cells was detected. The intensity of survivin staining was usually homogenous within a given case, but the number of positive tumor cells stained by the anti-survivin mAb varied between 20 and 100%, depending on the case investigated. After multiplying the weighted survivin score, 118 cases of breast carcinoma in this series were defined as positive (70.7%), with a weighted survivin score from 1 to 12. The distributions of weighted survivin scores were 0 for 49 cases, 1 for 17 cases, 2 for 26 cases, 3 for 30 cases, 4 for 14 cases, 6 for 13 cases, 8 for 8 cases, 9 for 7 cases, and 12 for 3 cases.

View larger version (129K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Histochemical staining for survivin and apoptotic tumor cells. a, survivin expression was restricted to the cytoplasm of tumor cells (arrows; x400). b, in situ labeling for apoptosis in a similar area detected only one apoptotic tumor cell (arrow; x400).

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. a, Kaplan-Meier curves for overall survival rates of patients with breast carcinoma categorized according to survivin expression. No significant difference was found between the groups (P = 0.108; log-rank test). b, Kaplan-Meier curves for overall survival rates of patients with breast carcinoma categorized by high versus low AI (P = 0.028; log-rank test).

	DISCUSSION

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We found compelling evidence that the presence of survivin in breast carcinoma was strongly associated with expression of bcl-2 and with reduced AI (Tables 2 and 3) . Our results agree with the findings of previous investigations, which showed a similar association between survivin and bcl-2 expression in neuroblastoma (25) , gastric cancer (11) , colorectal cancer (12) , and high-grade non-Hodgkin’s lymphoma.³ The percentage of bcl-2-positive cases in our series of breast cancer (76.0%) was comparable with those reported by other investigators (26) . The accumulation of mutated p53 protein was detected in 23.6% of the present series of patients. The rate of p53 accumulation was within the range of values reported by others (27, 28, 29) . The survivin gene is encoded at chromosome 17q25 (30) , whereas the bcl-2 gene is located at chromosome 18q21 and may be involved in the tumorigenic t(14;18) translocation (10) . These data imply that other transcriptional factors may contribute to the coregulation of both gene products in the progression of cancer. In this context, both survivin and bcl-2 genes are regulated by TATA-less, GC-rich promoter sequence in similar manners, and both are markedly transcribed in actively proliferating cell types (9) , suggesting common mechanism(s) of transcriptional activation. However, regardless of the pathway of simultaneous coexpression, it appears that survivin and bcl-2 proteins may mediate nonoverlapping, antiapoptosis mechanisms. Although bcl-2 is an integral inner mitochondrial membrane protein implicated in counteracting cytochrome c release from the mitochondria, IAP molecules, potentially including survivin, prevent apoptosis by targeting the terminal effectors caspase-3 and caspase-7 (8 , 31 , 32) . Survivin is expressed in the G₂-M phase of the cell cycle in a cell cycle-regulated manner and associates with microtubules of the mitotic spindle. Disruption of survivin-microtubule interactions results in loss of survivin’s antiapoptosis function and increased caspase-3 activity during mitosis. The overexpression of survivin in cancer may obliterate this apoptotic check point and allow aberrant progression of transformed cells through mitosis (32) . In breast carcinoma and in many of the most common human cancers, inhibition of apoptosis may be a general feature, and expression of survivin alone or survivin plus other antiapoptosis genes like bcl-2 may cause more pronounced antiapoptotic effects, as reflected in the significantly reduced apoptotic index observed in our series.

Although decreased AI associated with survivin expression was shown to be an indicator of poor prognosis in breast carcinoma, the predictive value was not as strong as was observed in colorectal carcinoma (P = 0.024 versus P = 0.0001; Ref. 12 ). One of the factors relevant to this issue may be the limited number of patients studied in this trial and the various responses to chemotherapy and/or endocrine therapy (case by case) that were given postoperatively to the present series of patients. Clark et al. (33 , 34) has recently proved that either mitosin expression or telomerase activity, a regulator of S-phase fraction, is an independent prognostic factor in node-negative and node-positive breast cancers. Therefore, identification of prognostic significance of apoptotic index should be clarified with additional use of these prognostic markers. bcl-2 has been reported to be frequently expressed in breast cancer and to be associated with positivity for ERs in both node-negative and node-positive breast cancers (35 , 36) . These findings are in agreement with our finding of a significant positive relationship between bcl-2 immunoreactivity and ER status (P = 0.024). However, unlike survivin proteins, the association of bcl-2 with a favorable clinical prognosis in breast cancer is reported (26 , 36) . One explanation for this paradox is that bcl-2 may have still unrecognized and other nonapoptotic functions. In clinical studies, bcl-2 expression is inversely correlated with S-phase fraction and tumor size in breast cancer (37 , 38) . Furthermore, bcl-2 has proved to have potential to lead to prolongation of the cell cycle as well as a decrease in vitro breast cancer growth (39) .

It is well known that there are multiple genetic pathways that control apoptosis, a part of which is probably regulated by survivin and/or bcl-2. Therefore, changing the level of expression of these proteins may not necessarily have an effect on outcome of therapy. A recent in vitro study demonstrated, however, that antisense survivin RNA down-regulated expression of endogenous survivin in transformed cells and resulted in increased apoptotic cell death (32) . In this context, in addition to chemotherapy and endocrine therapy, targeted antagonists of survivin may be beneficial as apoptosis-based therapy for breast carcinoma.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Dario C. Altieri (Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, New Haven, CT) for providing the anti-survivin antibody (8E2).

	FOOTNOTES

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

¹ To whom requests for reprints should be addressed, at Department of General and Gastroenterological Surgery, Osaka Medical College, 2-7 Daigaku-Machi, Takatsuki City, Osaka 569-8686, Japan.

² The abbreviations used are: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; AI, apoptotic index; ER, estrogen receptor; mAb, monoclonal antibody.

³ Unpublished observations.

Received 6/22/99; revised 9/23/99; accepted 9/27/99.

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				R. Kaneko, N. Tsuji, K. Asanuma, H. Tanabe, D. Kobayashi, and N. Watanabe Survivin Down-regulation Plays a Crucial Role in 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Inhibitor-induced Apoptosis in Cancer J. Biol. Chem., July 6, 2007; 282(27): 19273 - 19281. [Abstract] [Full Text] [PDF]

				S. Pina-Oviedo, K. Urbanska, S. Radhakrishnan, T. Sweet, K. Reiss, K. Khalili, and L. Del Valle Effects of JC Virus Infection on Anti-Apoptotic Protein Survivin in Progressive Multifocal Leukoencephalopathy Am. J. Pathol., April 1, 2007; 170(4): 1291 - 1304. [Abstract] [Full Text] [PDF]

				P. Kumar, I. K. Coltas, B. Kumar, D. B. Chepeha, C. R. Bradford, and P. J. Polverini Bcl-2 Protects Endothelial Cells against {gamma}-Radiation via a Raf-MEK-ERK-Survivin Signaling Pathway That Is Independent of Cytochrome c Release Cancer Res., February 1, 2007; 67(3): 1193 - 1202. [Abstract] [Full Text] [PDF]

				X.-H. Peng, P. Karna, R. M. O'Regan, X. Liu, R. Naithani, R. M. Moriarty, W. C. Wood, H.-Y. Lee, and L. Yang Down-Regulation of Inhibitor of Apoptosis Proteins by Deguelin Selectively Induces Apoptosis in Breast Cancer Cells Mol. Pharmacol., January 1, 2007; 71(1): 101 - 111. [Abstract] [Full Text] [PDF]

				N. Cosgrave, A. D K Hill, and L. S Young Growth factor-dependent regulation of survivin by c-myc in human breast cancer J. Mol. Endocrinol., December 1, 2006; 37(3): 377 - 390. [Abstract] [Full Text] [PDF]

				X.-H. Peng, P. Karna, Z. Cao, B.-H. Jiang, M. Zhou, and L. Yang Cross-talk between Epidermal Growth Factor Receptor and Hypoxia-inducible Factor-1{alpha} Signal Pathways Increases Resistance to Apoptosis by Up-regulating Survivin Gene Expression J. Biol. Chem., September 8, 2006; 281(36): 25903 - 25914. [Abstract] [Full Text] [PDF]

				P. N. Span, V. C.G. Tjan-Heijnen, J. J.T.M. Heuvel, J. B. de Kok, J. A. Foekens, and F. C.G.J. Sweep Do the Survivin (BIRC5) Splice Variants Modulate or Add to the Prognostic Value of Total Survivin in Breast Cancer? Clin. Chem., September 1, 2006; 52(9): 1693 - 1700. [Abstract] [Full Text] [PDF]

				S. Fukuda and L. M. Pelus Survivin, a cancer target with an emerging role in normal adult tissues Mol. Cancer Ther., May 1, 2006; 5(5): 1087 - 1098. [Abstract] [Full Text] [PDF]

				B. M. Ryan, G. E. Konecny, S. Kahlert, H.-J. Wang, M. Untch, G. Meng, M. D. Pegram, K. C. Podratz, J. Crown, D. J. Slamon, et al. Survivin expression in breast cancer predicts clinical outcome and is associated with HER2, VEGF, urokinase plasminogen activator and PAI-1 Ann. Onc., April 1, 2006; 17(4): 597 - 604. [Abstract] [Full Text] [PDF]

				W. Xia, J. Bisi, J. Strum, L. Liu, K. Carrick, K. M. Graham, A. L. Treece, M. A. Hardwicke, M. Dush, Q. Liao, et al. Regulation of Survivin by ErbB2 Signaling: Therapeutic Implications for ErbB2-Overexpressing Breast Cancers Cancer Res., February 1, 2006; 66(3): 1640 - 1647. [Abstract] [Full Text] [PDF]

				T. Gritsko, A. Williams, J. Turkson, S. Kaneko, T. Bowman, M. Huang, S. Nam, I. Eweis, N. Diaz, D. Sullivan, et al. Persistent Activation of Stat3 Signaling Induces Survivin Gene Expression and Confers Resistance to Apoptosis in Human Breast Cancer Cells Clin. Cancer Res., January 1, 2006; 12(1): 11 - 19. [Abstract] [Full Text] [PDF]

				H. Asanuma, T. Torigoe, K. Kamiguchi, Y. Hirohashi, T. Ohmura, K. Hirata, M. Sato, and N. Sato Survivin Expression Is Regulated by Coexpression of Human Epidermal Growth Factor Receptor 2 and Epidermal Growth Factor Receptor via Phosphatidylinositol 3-Kinase/AKT Signaling Pathway in Breast Cancer Cells Cancer Res., December 1, 2005; 65(23): 11018 - 11025. [Abstract] [Full Text] [PDF]

				J. G. Hodgson, T. Malek, S. Bornstein, S. Hariono, D. G. Ginzinger, W. J. Muller, and J. W. Gray Copy Number Aberrations in Mouse Breast Tumors Reveal Loci and Genes Important in Tumorigenic Receptor Tyrosine Kinase Signaling Cancer Res., November 1, 2005; 65(21): 9695 - 9704. [Abstract] [Full Text] [PDF]

				Y. Fujie, H. Yamamoto, C. Y. Ngan, A. Takagi, T. Hayashi, R. Suzuki, K. Ezumi, I. Takemasa, M. Ikeda, M. Sekimoto, et al. Oxaliplatin, a Potent Inhibitor of Survivin, Enhances Paclitaxel-induced Apoptosis and Mitotic Catastrophe in Colon Cancer Cells Jpn. J. Clin. Oncol., August 1, 2005; 35(8): 453 - 463. [Abstract] [Full Text] [PDF]

				D. A. Fennell Caspase Regulation in Non-Small Cell Lung Cancer and its Potential for Therapeutic Exploitation Clin. Cancer Res., March 15, 2005; 11(6): 2097 - 2105. [Abstract] [Full Text] [PDF]

				X.-H. Peng, Z.-H. Cao, J.-T. Xia, G. W. Carlson, M. M. Lewis, W. C. Wood, and L. Yang Real-time Detection of Gene Expression in Cancer Cells Using Molecular Beacon Imaging: New Strategies for Cancer Research Cancer Res., March 1, 2005; 65(5): 1909 - 1917. [Abstract] [Full Text] [PDF]

				S. Idenoue, Y. Hirohashi, T. Torigoe, Y. Sato, Y. Tamura, H. Hariu, M. Yamamoto, T. Kurotaki, T. Tsuruma, H. Asanuma, et al. A Potent Immunogenic General Cancer Vaccine That Targets Survivin, an Inhibitor of Apoptosis Proteins Clin. Cancer Res., February 15, 2005; 11(4): 1474 - 1482. [Abstract] [Full Text] [PDF]