J Cancer Res Clin Oncol (2005) 131: 504–510 DOI 10.1007/s00432-005-0682-z

O R I GI N A L P A P E R

T. Ponnelle Æ C. Chapusot Æ L. Martin Æ A. M. Bouvier S. Plenchette Æ J. Faivre Æ E. Solary Æ F. Piard

Cellular localisation of Survivin: impact on the prognosis in colorectal cancer

Received: 22 May 2004 / Accepted: 17 February 2005 / Published online: 18 May 2005
Springer-Verlag 2005

Abstract Purpose: The present study was designed to determine whether the nuclear or cytoplasmic expression of survivin, was related to clinicopathological parameters and survival in sporadic colon carcinomas. Methods: Western blotting of cell fractions and immunocytochemical methodology were used in five human colon cancer cell lines. Immunohistochemical study was performed in formalin-fixed paraffin-embedded section from 46 patients with sporadic colorectal adenocarcinomas with a polyclonal antibody directed against survivin. Apoptotic index was evaluated by using the M30 antibody. Survival rates were estimated by the Kaplan– Meier method and compared using the log-rank test. Multivariate survival analysis was performed by the Cox proportional hazards model. Results: Western blotting and immunocytochemistry analyses confirmed that survivin could be detected both in the nucleus and the cytoplasm. Immunohistochemical analysis demonstrated that 39% of tumours expressed survivin in the nucleus and 41% in the cytoplasm. No relationship was observed between survivin expression and clinicopathological features. Unexpectedly, the apoptotic index appeared to be linked with high survivin nuclear expression. Overall, 3-year observed survival rate was 73% in patients with cytoplasmic survivin expression versus 48% for negative expression (P=0.14). Survival was 72% versus 50% for positive nuclear survivin T. Ponnelle Æ C. Chapusot Æ L. Martin Æ F. Piard Faculte´ de Me´dicine, Service d’anatomie et de Cytologie Pathologiques, 7, boulevard Jeanne d’Arc, BP 87900, 21079 Dijon Cedex, France T. Ponnelle (&) Æ C. Chapusot Æ A. M. Bouvier Æ J. Faivre F. Piard Registre Bourguignon des Cancers Digestifs, INSERM EMI 01-06, 21079 Dijon Cedex, France E-mail: [email protected] Tel.: +33-3-80393344 Fax: +33-3-80393350 S. Plenchette Æ E. Solary Faculty of Medicine, INSERM U-517, IFR 100, 21079 Dijon Cedex, France

expression versus negative (P=0.16). After adjustment for age and stage, cytoplasmic survivin expression was a significant prognostic factor. A high level of expression was associated to a better survival: RR=0.35 [0.13– 0.98], P=0.045. Conclusion: These results indicate that the analysis of the subcellular expression of survivin is a determining factor to define the prognostic value. Its evaluation, using a polyclonal antibody, might help clinicians in the stratification of patients with colorectal cancer. Keywords Survivin Æ Inhibitor of apoptosis protein (IAP) Æ Apoptosis Æ Colorectal cancer Æ Prognosis

Introduction Colorectal cancer is the second leading cause of cancerrelated mortality in Western Countries. Tumour kinetic studies suggest that tumour growth does not only result from increased rates of cell proliferation but also from decreased rates of apoptosis. Survivin belongs to the inhibitor of apoptosis (IAP) gene family which encodes a group of structurally related proteins that are involved in an increasing number of seemingly unrelated cellular functions (Salvesen et al. 2002). These proteins include XIAP, c-IAP1, c-IAP2 and survivin that negatively interfere with apoptosis, supposedly due to their ability to inhibit a limited number of caspases, mainly caspase-3, 7 and 9. These IAPs are highly evolutionary conserved proteins that are defined by the presence of at least one Baculovirus IAP repeat (BIR) domain in their primary structure (Uren et al. 1998). Unlike c-IAP1 and c-IAP2, survivin contains a single BIR domain and lacks a carboxy-terminal RING finger. This protein directly inhibits caspase-3 and caspase-7 activity but also regulates the G2/M phase of the cell cycle by associating with mitotic spindle microtubules. Survivin is expressed during foetal development and its

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expression is down-regulated in most terminally differentiated cells (Adida et al. 1998). However, the protein is expressed again in a number of transformed cell types (Ambrosini et al. 1997) and its expression positively correlates with proliferation and angiogenesis (Kawasaki et al. 2001). Survivin expression was shown to predict poor prognosis and shorter survival in various human cancers (Adida et al. 2000; Ikeguchi et al. 2002a, 2002c; Ikehara et al. 2002; Kappler et al. 2003; Kato et al. 2001; Kawasaki et al. 1998; Ku et al. 2004; Monzo et al. 1999; Sarela et al. 2000, 2001) and was proposed as a new target for disrupting cell survival pathways in cancer cells (Ambrosini et al. 1998). The link between high expression of survivin and poor prognosis has recently been called into question in gastric, breast, bladder cancer and osteosarcoma (Kennedy et al. 2003; Lehner et al. 2002; Okada et al. 2001; Trieb et al. 2003). The aim of this study was to evaluate the expression of survivin in five human colonic cancer cell lines and in a series of 46 colorectal carcinomas, to determine the correlation between survivin expression and tumour apoptosis, and to assess any association between expression of this protein and survival.

Materials and methods Cell lines The human colonic carcinoma cell lines HT 29 (mutated p53), HCT 116 (wild-type p53), SW 480 and SW 620 (mutated p53) were obtained from ATCC (Rockville, MD, USA). The HCT8R cell line (wild-type p53) was kindly provided by Marc Mareel (Gend, Belgium). These colonic cancer cell lines were maintained in Eagle’s minimum essential medium (EMEM) complemented with 10% foetal calf serum (GibcoBRL, Life Technologies, Cergy Pontoise, France) and 2 mM Lglutamine (Biowhitaker Co., Fontenay sous Bois, France). They were cultured in monolayer at 37C in an atmosphere of 5% CO2. Patients and samples Forty-six consecutive patients in whom surgical resection of a primary sporadic colorectal cancer was performed at Dijon University Hospital between January 1998 and March 1999 were included in this study. Tumour and mucosa samples were embedded in paraffin after 16 h formalin fixation. None of the patients (16 males, 30 females, mean age 74±13.5 years, range 27– 95) had received any anticancer treatment prior to surgery. According to the TNM classification, 31 cancers were stage I or II and 15 were stage III or IV. Thirty-one were left-sided tumours (distal to splenic flexure) and fifteen were right-sided. Forty-two were well to moderately differentiated adenocarcinomas and four were poorly differentiated. Thirty-seven cancers displayed a

common inflammatory stromal lymphoid infiltrate, whereas nine carcinomas showed a strong lymphoid infiltrate including lymphoid follicles with germinal centres. At the time of analysis, mean follow-up was 35 months (range 28–42) and 16 patients (35%) died from disease progression. Cell lysate preparation Whole cell lysates were prepared by lysing the cells in boiling buffer (1% SDS, 1 mM Na-vanadate, 10 mM Tris, pH 7.4) in the presence of protease inhibitors [0.1 mM phenylmethylsulfonylfluoride (PMSF), 2.5 lg/ ml pepstatin, 10 lg/ml aprotinin, 5 lg/ml leupeptine], then reducing the sample viscosity by several passages through a 26-gauge needle. For fractionation, cells were resuspended in ice cold buffer [sucrose 250 mM, HEPES 20 mM, KCl 10 mM, MgCl2 1.5 mM, EDTA 1 mM, EGTA 1 mM, DTT 1 mM, PMSF 17 lg/ml, aprotinin 8 lg/ml, leupeptine 2 lg/ml (pH 7.4)] before passing them through an ice-cold cell homogenizer. Non-lysed cells and nuclei were pelleted via a 10 min 750 g spin. This step was repeated twice and the supernatant was centrifuged at 10,000 g for 25 min before collecting the resulting pellet corresponding to the mitochondria fraction. The other fraction was further centrifuged at 100,000 g for 1 h to collect the supernatant that represented the cytosolic fraction. Western blot analysis Protein concentration was measured by using the BioRad DC protein assay kit (Ivry sur Seine, France). Thirty micrograms of protein were incubated in loading buffer (125 mM Tris–HCl, pH 6.8, 10% ß-mercaptoethanol, 4.6% SDS, 20% glycerol and 0.003% bromophenol blue), separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and electroblotted to PVDF membrane (BioRad). After blocking non-specific binding sites overnight by 5% nonfat milk in TPBS (PBS, Tween 20 0.1%), the membrane was incubated for 2 h at room temperature with primary antibody. After two washes in TPBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PE) for 30 min at room temperature, then washed twice in TPBS. Immunoblot was revealed by autoradiography using enhanced chemoluminescence detection kit (Amersham, Les Ulis, France). Immunocytochemistry–immunohistochemistry Analysis of cell lines was performed on 5 lm thick sections of formalin-fixed and paraffin-embedded fibrinclot preparations. Analysis of colorectal carcinomas was performed on 5 lm thick sections of 10% formalin-fixed

506

embedded samples. A Supersensitive Immunodetection System (Biogenex, San Ramon, CA, USA) and the Amino Ethyl Carbazol chromogen were used for both immunocyto and immunohistochemistry. Slides were autoclaved in 10 mM citrate buffer (pH 6) for 10 min to allow antigen retrieval before a 12-h incubation at 4C with primary antibodies directed against survivin (1/600, polyclonal, NB500–201, Novus biological) and cleaved cytokeratin 18 (1/100, monoclonal, M30 Cytodeath, Roche). Survivin expression was estimated semi-quantitatively with a score including the percentage of cells displaying a cytoplasmic or nuclear signal. The mean percentage of positive tumour cells was determined in ten areas at high magnification (·400) and was graded from 0 to 4 (0=<5% positive cells; 1=6–25%; 2=26– 50%; 3=51–75%; 4=76–100%). Apoptotic tumour cells were identified using the M30 antibody that specifically recognizes a caspase-cleaved cytokeratin 18. The average number of tumour cells stained by M30 Cytodeath per field at ·100 was evaluated in at least five nonnecrotic areas. Because of the lack of reference data, patients were arbitrarily divided into two groups according to the level of survivin expression closest to the median. Nuclear and cytoplasmic survivin expression was considered as positive when more than 25% and more than 5% of tumour cells were stained respectively. The same method was used for the apoptotic index. Tumours with more than ten positive cells per field at ·100 magnification stained by M30 Cytodeath were considered as having a high apoptotic index. Negative controls were obtained by omitting the primary antibody. The normal mucosa of each sample was analysed simultaneously and was used as an internal positive control. Slides were read by two observers without knowledge of the clinical data. Statistical analysis Chi-square tests and Fisher exact tests were used to compare categorical data. Survival probability analyses were performed using the Kaplan–Meier method and were calculated from the date of diagnosis to the date of death or most recent follow-up. Statistical significance was assessed by the log-rank test. Independent predictive effect for the survival of cytoplasmic and nuclear survivin expression was determined using the Cox proportional method. Each model was adjusted for age and stage at diagnosis. All data were analysed by STATA statistical software.

Results Expression of survivin in colon cancer cell lines Western blot analysis showed that survivin was detected in all five cell lines and that its expression varied slightly from one cell line to another (Fig. 1a). Immunoblot

Fig. 1 a Western blotting for survivin in cultured colonic cell lines. In all cases, lysates were normalized for total protein content (50 lg) before SDS-PAGE/immunoblot assay. Single bands of various intensity around 16 kDa indicated the expression of survivin at various levels. b Fractionated Western blotting for survivin in colonic cancer cell line (HT 29). The protein was mainly expressed in the nucleus and cytosol

analysis of subcellular fractions carried out in HT-29 cells indicated that the expression of survivin was greater in the nucleus than in the cytosol. The protein was not associated with the mitochondria (Fig. 1b). Immunocytochemical analysis confirmed that survivin could be expressed both in the nucleus and in the cytoplasm (Fig. 2a). Expression of survivin in human normal colon mucosa Then we performed an immunohistochemical analysis of the expression of survivin in samples of normal colon mucosa. Survivin was expressed in the nucleus of epithelial cells scattered at the bottom of the colon crypts, whereas cytoplasmic survivin was diffusely expressed along the crypt with intensity slightly decreasing from the bottom to the top (Fig. 2b). Expression of survivin studied protein in human colon tumour samples The same immunohistochemical method was used to analyse the expression of survivin in 46 samples of human colon cancers. Tumour cells demonstrated a nuclear and/or a cytoplasmic granular staining pattern (Fig. 2c). The intensity of the signal was usually homogeneous within a given case. The rate of stained tumour cells varied from 1% to 100%, depending on the tumour. The percentage of tumours with nuclear and/or cytoplasmic staining, according to the threshold values defined in the material and methods section are of 39% and 41%, respectively. The cleaved cytokeratin 18 staining was exclusively cytoplasmic. The average number of tumour cells stained by M30 Cytodeath varied between 1 and 400 cells per field. Twenty cases (43%) had a low apoptotic index.

507 Fig. 2 a Immunocytochemistry for survivin of cultured colonic cancer cells (HT 29). Both cytoplasmic and nuclear signal was observed in most of tumour cells. b Immunohistochemistry of normal colon mucosa for survivin showing a variation of nuclear and cytoplasmic expression of the protein from the bottom to the top of the crypts. c Immunohistochemistry for survivin in colorectal cancer: well-balanced nuclear and cytoplasmic expression

Correlations between expression of survivin, clinicopathological factors and apoptotic index There was little correlation between either survivin expression or apoptotic index and the clinicopathological characteristics of patients (Table 1). A high apoptotic index was associated with proximal location of the tumour (P=0.005). An elevated apoptotic index was related to a nuclear localization of survivin (P=0.032) (Table 2). Survival analysis The overall 3-year observed survival rate was 73% in patients with cytoplasmic survivin expression versus

48% for negative survivin expression. Survival was 72% versus 50% for positive nuclear survivin expression versus negative. The logrank test comparing survival curves (Fig. 3.) according to cytoplasmic or nuclear survivin expression was not significant, respectively 0.14 and 0.16. Impact on prognosis according to the nuclear or cytoplasmic expression of the survivin was evaluated in two multivariate analyses (Table 3). After adjusting for age and stage at diagnosis, the relative risk of death was lower for cytoplasmic (RR= 0.35, P=0.045) and nuclear (RR=0.45) survivin expression. There was no significant link between the apoptotic index assessed by the M30 Cytodeath antibody and patient survival in this series.

508 Table 1 Proportion of cancers with high apoptotic index or with survivin expression according to clinicopathological factors

N nucleus, C cytoplasm

High apoptotic index (%)

n

Age <75 years 22 >75 years 24 Sex Male 16 Female 30 TNM stage I and II 31 III and IV 15 Tumour location Proximal 15 Distal 31 Tumour differentiation Well and mod 42 Poor 4 Lymphoid response Strong 9 Sparse 37

P

Survivin N (%)

P

C (%)

P

54 58

0.796

36 56

27 0.713

54

0.064

50 60

0.515

44 36

25 0.639

50

0.126

61 47

0.348

45 27

0.336

48 27

0.210

87 42

0.005

40 39

0.933

47 38

0.607

55 75

0.622

38 50

0.639

40 50

0.712

55 57

0.948

67 32

0.124

33 43

0.716

Discussion The prognostic value of survivin expression in colorectal cancer cells remains a controversial issue. Most of the studies analysing the prognostic significance of survivin expression were performed without taking into account the subcellular location of the protein, or reported survivin location restricted to the cytoplasm of tumour cells (Adida et al. 2000; Kawasaki et al. 1998; Lu et al. 1998; Sarela et al. 2000, 2001; Tanaka et al. 2000). Our study demonstrates that survivin can present both nuclear and cytoplasmic expression in cell lines and in tumour samples. In addition, it suggests that the subcellular location of survivin could influence the prognostic significance of its expression in human sporadic colon cancers. Survivin is widely expressed in foetal tissues and in human cancers, but was generally not found, or found at low level in normal adult tissues (Deveraux et al. 1999). In a global gene expression analysis, survivin was identified as one of the top genes invariably up-regulated in many types of cancer as compared with normal tissues (Velculescu et al. 1999). Thus, overexpression of survivin, which is an intriguing interface between apoptosis and cell-cycle progression, may provide a survival advantage for tumour progression (Thompson 1995).

The critical question of what may cause re-expression of survivin in cancer remains largely unanswered but a complex response to dedifferentiation of normal epithelium appears likely (Adida et al. 1998). Survivin is the

Table 2 Proportion of cancers with high apoptotic index according to survivin expression High apoptotic index Percentage Nuclear survivin + 78 43 Cytoplasmic survivin + 58 56

P n 14/18 12/28

0.032

11/19 15/27

0.88

Fig. 3 Kaplan–Meier survival curves for positive and negative cases of colon cancer regarding survivin nuclear and cytoplasmic expression

509 Table 3 Relative risk of death 3 years from diagnosis according to survivin expression (Cox model after adjustment for age and stage at diagnosis) RR

95%CI

P

0.13–0.98

0.045

0.17 –1.20

0.114

Survivin cytoplasmic +

1 0.35

Survivin nuclear +

1 0.45

only member of the IAP family for which a function in the nucleus was identified (Suzuki et al. 2000). This protein is overexpressed in the G2/M phase of the cell cycle and binds to the microtubules of the mitotic spindle (Li et al. 1998). Survivin assembly on the mitotic apparatus seems to be required for its antiapoptotic function. It has been hypothesized that, in cancer cells, overexpressed survivin suppressed an apoptotic checkpoint during mitosis (Ambrosini et al. 1998) and promoted aberrant progression through mitosis (Li et al. 1998). Fortugno et al. (2002) reported immunochemically distinct nuclear and cytoplasmic pools of survivin involved in the regulation of mitotic spindle microtubules formation and function. Thus, Mahotka et al. (2002) described differential subcellular localization of functionally divergent survivin splice variants. They show that the non-antiapoptotic survivin-2B co-localize with the survivin mainly in the cytoplasm, whereas the antiapoptotic isoform survivin-dEx3 is preferentially found in the nucleus. Most retrospective trials associated survivin RNA level or cytoplasmic expression of the protein with a poor outcome in various cancers (Adida et al. 2000; Azuhata et al. 2001; Ikeguchi et al. 2002b; Kato et al. 2001; Kawasaki et al. 1998; Ku et al. 2004; Monzo et al. 1999; Sarela et al. 2000; Sui et al. 2002). However, some recent series dealing with the nuclear expression of survivin underlined the relation between a nuclear immunohistochemical detection and a better survival in gastric, breast, bladder cancers and osteosarcoma (Kennedy et al. 2003; Lehner et al. 2002; Okada et al. 2001; Trieb et al. 2003). Furthermore, in those reports, the detection of the protein in tumour cell cytoplasm did demonstrate a similar trend. In our series, both the nuclear and the cytoplasmic expression of survivin demonstrated a favourable influence upon survival with a statistically significant difference for cytoplasmic expression. The different results between previous and recent studies could be explained by the choice of the antibodies. Whereas studies carried out with the monoclonal antibody (clone 8E2) associated survivin expression with a poor prognosis, our study and those performed in gastric, breast, bladder cancers and osteosarcoma were performed with a polyclonal antibody. Unlike the monoclonal antibody that only detects one or

a few cytoplasmic isoforms of survivin, the polyclonal antibodies are able to identify several of the recently described isoforms of survivin (Mahotka et al. 1999), including those that are localized in the nucleus (Mahotka et al. 2002; Fortugno et al. 2002). The survivin-2B, which lost anti-apoptotic potential, may act as a naturally occurring antagonist of survivin (Islam et al. 2000; Mahotka et al. 1999). Recently, a quantitative reverse transcriptase polymerase chain reaction study demonstrated a significant stage-dependent decrease in the expression of survivin-2B in gastric carcinoma (Krieg et al. 2002). In our study, the best patient survival rate could be linked to the nuclear and cytoplasmic expression of a non-antiapoptotic isoform. Unfortunately, no antibodies are currently available to detect specifically these novel splice variants. In our study, the rate of apoptotic malignant cells was quantified with the monoclonal antibody M30 that recognizes a caspase-cleaved fragment of cytokeratin 18. Previous reports have shown that cytoplasmic expression of survivin assessed by the monoclonal antibody 8E2 was related to a low apoptotic index in various tumours (Kawasaki et al. 2001; Lu et al. 1998). Conversely, we observed that a high apoptotic index was associated with the nuclear expression of survivin. As previously stated, it can be explained by the ability of our antibody to detect a nuclear proapoptotic (or nonantiapoptotic) isoform of the survivin. Nevertheless, we were unable to confirm the previously reported prognostic significance of this index (Kawasaki et al. 1998). As previously shown, we found that proximal location of the tumour was associated with high apoptotic index (Jonges et al. 2001; Zhang et al. 1999). The prognostic value of survivin expression in colorectal cancer remains a controversial issue. Our study demonstrates that overexpression of survivin in the cytoplasm and nucleus of tumour cells is indicative of improved survival and suggests that the kind of the antibody could explain the contradictory reports of the literature. Acknowledgements This work was supported by grants of the ‘‘Ligue contre le cancer de Saoˆne et Loire ’’ and ‘‘Fondation pour la recherche me´dicale’’. We would like to thank David Rageot, Martine Goiset and Didier Carnet for technical help.

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