Survivin in Adrenocortical Tumors - Pathophysiological Implications and Therapeutic Potential
Authors
S. Sbiera1, M. Kroiss1, T. Thamm1, M. Beyer1, F. Majidi1, D. Kuehner1, M. Wobser2, J. C. Becker3, P. Adam4, C. Ronchi1, B. Allolio1, M. Fassnacht1, 5
Affiliations
Affiliation addresses are listed at the end of the article
Key words
adrenocortical carcinoma BIRC5 · apoptosis
· prognosis
· therapy
Abstract
▼
Treatment options for adrenocortical carcinoma (ACC) are very limited. In other solid tumors, small vaccination trials targeting the anti-apop- totic molecule survivin suggested immunological and clinical benefit in selected patients. There- fore, we investigated whether survivin might be a suitable target for immunotherapy in ACC. Sur- vivin mRNA and protein expression was assessed in adrenal tissue specimens [by real-time-PCR in 29 ACC, 24 adrenocortical adenomas (ACA) and 12 normal adrenal glands; by immunohis- tochemistry in 167 ACCs, 15 ACA, and 5 normal adrenal glands]. Expression was correlated with clinical outcome using Kaplan-Meier and Cox regression analyses. The anti-apoptotic role of survivin was investigated in the SW13 ACC cell line using survivin siRNA. The presence of spon- taneous survivin specific T-cells in peripheral
blood was assessed by FACS dextramere stain- ing in 29 ACC patients in comparison to healthy controls. Survivin mRNA in ACC was significantly overexpressed when compared with ACA or normal adrenal glands. Immunohistochemistry confirmed survivin protein expression in 97% of the ACCs. In 83% of samples, staining was mod- erate or high and clinical outcome in this sub- group showed a trend towards poorer prognosis [hazard ratio for death 2.28 (95% CI 0.99-5.28); p=0.053]. Survivin knockdown in SW-13 cell sig- nificantly increased the rate of apoptosis. Finally, spontaneous survivin-reactive T cells were detectable in 3 of 29 ACC patients. In conclu- sion, our data suggest that survivin could play an important role in the anti-apoptotic mechanisms in ACC and provide first hints that targeting sur- vivin might be an interesting new therapeutic approach in this rare disease.
received 27.07.2012 accepted 12.10.2012
Bibliography
DOI http://dx.doi.org/ 10.1055/s-0032-1327750 Published online: November 9, 2012 Horm Metab Res 2013; 45: 137-146 @ Georg Thieme Verlag KG Stuttgart . New York ISSN 0018-5043
Correspondence
M. Fassnacht, MD Department of Internal Medicine I Endocrine and Diabetic Unit University Hospital of Würzburg Oberdürrbacher Straße 6 97080 Würzburg Germany Tel .: +49/931/201 39021 Fax: +49/931/201 61632 fassnacht_m@medizin. uni-wuerzburg.de
Introduction ▼
Since almost 2 decades there is increasing evi- dence that survivin is involved in tumor cell resistance in a variety of human neoplasms [1]. Survivin, also known as baculovirus inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) is, as its name suggests, a member of the IAP family that plays a key role in regulation of apoptosis and cell division [2,3]. By inhibiting apoptosis and promoting mitosis survivin facili- tates cancer cell survival and growth [4]. High expression of survivin is often associated with a more aggressive tumor, drug, and radiotherapy resistance associated with reduced survival [5- 7]. Several preclinical studies have demonstrated that downregulation of survivin expression or function decreased tumor growth and sensitized the tumor cells to a series of chemotherapeutic drugs and radiation both in vitro and in vivo in
various types of human tumors [8-13]. In addi- tion, the detection of spontaneous circulating cytotoxic T-lymphocytes against survivin in patients with different types of tumors indicated the ability to raise an immune response against this protein [14-17].
Adrenocortical carcinoma (ACC) is a rare tumor with an incidence of under 2 new cases per mil- lion per year [18] with a poor prognosis [19,20]. The overall survival rates at 5 years in different series range between 35 and 50% [21-24]. The treatment of choice in early stages is radical sur- gery, but most patients experience disease relapse even after complete removal of the pri- mary tumor [25] (Erdogan et al. [26], unpublished results). Therefore, most but not all centers now recommend adjuvant mitotane after complete resection [19,27-31]. Very recently, the first ran- domized trial in ACC established now the combi- nation of mitotane with etoposide, doxorubicin,
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and cisplatin as standard chemotherapy regimen in advanced disease [32]. However, even this multi-drug regimen has an objective response rate below 25% and management of these patients is still challenging [33]. Poor clinical outcome is linked to limited knowledge of ACC pathogenesis as only a limited number of prognostic markers have been proposed [34-38]. In addition, only very few markers predicting response to therapy have been suggested [39,40]. However, many chemotherapeutic drugs act ultimately through induction of apoptosis, their reduced efficacy likely relating to their inability to induce apop- tosis in the ACC cells [41,42].
Until now the role of survivin in ACC has not been investigated. In this study, we evaluated survivin expression both at mRNA and protein level in adrenocortical tissues of healthy and patho- logical origin. In addition, we correlated survivin expression lev- els with the clinical outcome of the ACC patients. Moreover, the role of survivin was scrutinized in the context of other ACC prog- nostic factors. Furthermore, we screened for the presence of cir- culating spontaneous survivin specific cytotoxic T-cells as an indication of an existing immune response against this compo- nent of the tumor. Finally, we knocked survivin in ACC cells down to elucidate its role in the apoptosis of these cells and to investigate the possibility to sensitize the cells to chemothera- peutic drugs.
Materials and Methods
▼
Patients
Adrenocortical tumor tissue was collected from a total of 194 patients undergoing adrenalectomy due to adrenocortical carci- noma (ACC, n=163) or adenomas (n=31) such as aldosterone- producing (APA) (n=9), cortisol-producing (CPA) (n=13), or endocrine inactive (EIA) (n=9) adenomas. Normal adrenal gland tissue samples from 16 patients undergoing nephrectomies were also included. In addition, blood samples were collected from 30 ACC patients, 3 patients with cortisol excess due to CPA and 9 healthy donors. The diagnosis of ACC was based on estab- lished clinical, biochemical and morphological criteria, and all histological diagnoses, including Weiss score and Ki67 expres- sion, were confirmed by the reference pathologist of the German ACC study group (Wolfgang Saeger, Hamburg).
Clinical data of the ACC patients, including follow-up and sur- vival data, were collected in a structured manner by the German ACC Registry (www.nebennierenkarzinom.de). For this study the follow-up information was locked as of January 2012. Tumor staging at the time of diagnosis was based on imaging studies, corroborated by the findings during surgery and recorded according to the ENSAT classification [23]. Surgical resection of the primary tumor was considered complete (R0 resection) if surgical, pathological and imaging reports did not show any evi- dence of a remaining disease. Presence of distant metastases was evaluated at the time of diagnosis and during follow-up vis- its by computerized tomography of chest and abdomen. Patients gave informed consent for collecting tissue and clinical data, the study was approved by the ethics committee of the University of Würzburg.
Survivin transcription levels
Tumor RNA/cDNA
A total of 65 tissue samples (29 primary ACC, 8 CPA, 8 APA, 8 EIA, and 12 normal adrenal glands) were shock-frozen immediately
after tissue removal using liquid nitrogen. Patients’ characteris- tics are described in . Table 1 (Part A). In addition, the 2 most often used ACC cell lines, NCI h-295 [43] and SW-13 [44] served as positive control. The cell cultures have been maintained as published before [45]. Total-RNA was extracted using the RNe- asy Mini Kit® (Quiagen, Hilden, Germany) from 30mg of each of the tissues following manufacturers instructions and immedi- ately converted into cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Munich, Germany) and cDNA was stored in aliquots at - 20℃.
Quantitative real-time PCR
To quantify survivin expression in normal adrenal glands and adrenal tumors, real-time PCR was performed on 500 ng from each of the cDNAs using TaqMan® Gene Expression Assay (Applied Biosystems, Foster City, CA, USA) and an iCycler (Bio- Rad Laboratories, Munich, Germany). The TaqMan® probes Hs00153353_m1 for survivin and Hs99999901_s1 for the 18S RNA loading control were used. The following protocol was per- formed: 50℃ for 2 min, 95℃ for 10 min and 45 cycles of 95 ℃ for 15 s and 60℃ for 60 s. Survivin expression levels were stand- ardized to the levels of ribosomal RNA.
Survivin protein expression Tissue array
Tissue from a total of 167 formalin-fixed, paraffin-embedded, adrenocortical carcinomas (134 primary tumors, 19 local recur- rences and 14 metastases), 5 from each CPA, APA and EIA and 5 normal adrenal glands (· Table 1, Part B) was assembled into 3 tissue microarrays (TMAs) as previously described [46].
Immunohistochemistry
The TMAs were deparaffinized in xylene and rehydrated in increasing dilutions of ethanol. An antigen retrieval step of 40 min with DakoCytomation Retrieval Solution (Dako, Glostrup, Denmark) in a high temperature vapor bath was performed and the endogenous peroxidase activity was quenched with Peroxi- dase Blocking Reagent (Dako, Glosdrup, Denmark) for 30 min at room temperature. Subsequently, the unspecific binding was blocked for 15 min at 37℃ using 20% human AB serum (Sigma- Aldrich, Steinheim, Germany, Lot. 017K0443) in phosphate buffer saline (PBS). The TMAs were then incubated for 1h at 37°℃ with 5 µg/ml horse radish peroxidase (HRP) linked polyclo- nal rabbit anti-survivin antibody (Novus Biologicals, Littleton, USA) and in case of the negative control with N-Universal Nega- tive Control Rabbit antibody (Dako, Glosdrup, Denmark). The slides were then washed 3 times with PBS and the survivin bound antibody signal was developed using the EnVision+® Sys- tem Anti-rabbit (Dako, Glosdrup, Denmark). The color reaction was performed with NovaRED substrate kit (Vector, Burlingame, USA). After counterstaining with hematoxylin, the arrays were then analyzed independently by 2 investigators and the different staining intensities were scored semi-quantitatively (from 0=negative to 3 =highly positive). Only samples with at least 2 out of 5 evaluable tissue cores were assessed. In case of inter- investigator discrepancies, the results were double-checked by an independent observer. Negative tissue samples were re-ana- lyzed in full tissue sections.
In silico analysis of survivin mRNA expression
As an independent cohort we analyzed survivin mRNA expres- sion from the high density oligonucleotide array data published
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| A: Cohort for mRNA analysis by real-time PCR. | |||
|---|---|---|---|
| Mean Age (SD) | Sex (M/F) | Mean primary tumor size (SD) | |
| ACC | |||
| Primary tumor (n=29*) | 50 (17) | (9/18) | 11.6 (4) |
| ENSAT I (n=1) | 46 (0) | (0/1) | 3 (0) |
| ENSAT II (n= 15) | 48 (19) | (5/10) | 12.5 (4) |
| ENSAT III (n=2) | 34 (24) | (0/2) | 12 (1.4) |
| ENSAT IV (n=9) | 57 (12) | (4/5) | 11 (3.6) |
| Adenoma | 50 (15) | (4/4) | 1.7 (0.7) |
| Aldosterone-producing adenoma (n=8) | 40 (8) | (3/5) | 2.8 (1.1) |
| Cortisol-producing adenoma (n=8) | 65 (10) | (6/2) | 4 (3.2) |
| Inactive adenoma (n=8) | 53 (11) | (8/4) | – |
| Normal adrenal gland (n=12) | |||
| B: Cohort for protein analysis by immunohistochemistry. | |||
|---|---|---|---|
| Mean Age (SD) | Sex (M/F) | Mean primary tumor size (SD) | |
| ACC | |||
| Primary tumor (n=134 ** ) | 49 (16) | (48/86) | 12 (4.4) |
| ENSAT I (n= 5) | 54 (24) | (2/3) | 4.7 (0.3) |
| ENSAT II (n=48) | 48 (17) | (19/29) | 11.9 (4.5) |
| ENSAT III (n=41) | 53 (14) | (14/27) | 11.8 (3.6) |
| ENSAT IV (n=34) | 47 (18) | (11/23) | 13.4 (4.4) |
| Local recurrence (n= 19) | 46 (17) | (9/10) | – |
| Distant metastases (n= 14) | 44 (11) | (3/11) | – |
| Adenoma | 47 (14) | (2/3) | 1.8 (0.7) |
| Aldosterone-producing adenoma (n=5) | 48 (14) | (0/5) | 2.6 (0.6) |
| Cortisol-producing adenoma (n=5) | 67 (9) | (2/3) | 2 (0.8) |
| Inactive adenoma (n=5) Normal adrenal gland (n=4) | 62 (16) | (3/1) | – |
| C: Cohort analyzed for survivin-specific cytotoxic T cells. | ||||
|---|---|---|---|---|
| Mean age (SD) | Sex (M/F) | Tumor present at the time of blood sampling (Y/N) | HLA-A2 (+/-) | |
| ACC patients Patients (n=29) | 50 (14) | (13/16) | (20/9) | (29/0) |
| Adenoma patients Cortisol-producing adenoma (n=3) | 41 (15) | (2/1) | (0/3) | (3/0) |
| Normal adrenal gland (n=9) | 32 (14) | (4/5) | – | (6/3) |
*In 2 cases no information is available
** In 6 cases tumor stage was not determined (SD)= standard deviation
by Giordano et al. [47]. Survivin expression data was extracted from the complete array data deposited by the authors in National Center for Biotechnology Information’s gene Expres- sion Omnibus under the accession number GSE10927. The array comprises normalized expression data from 10 normal adrenal glands, 22 adrenocortical adenomas, and 33 ACC originating from expression microarray analysis. For the 33 ACC samples we also used the hierarchical clustering information based on the principal component analysis the authors report as correlating with both the tumor grade and with patient survival data. Ana- lyzing the cluster distribution of survivin mRNA expression we determined the association of survivin expression with ACC patient survival.
Functional analyses of survivin in ACC cell-lines siRNA transfection
The ACC cell-line SW-13 was cultured in Leibowitz L-15 medium (Sigma-Aldrich, Steinheim, Germany) with 10% fetal calf serum (PAN Biotech, Aidenbach, Germany) and 2 mM L-glutamine (Inv- itrogen Corporation, Carlsbad, USA). The cells were transfected with 5nM survivin siRNA SI00299453 (Qiagen, Hilden, Ger- many) by electroporation for 10 milliseconds at 340V using a
square-wave protocol in a GenePulser Xcell instrument (BioRad Laboratories, Hercules, USA). To assess the transfection effi- ciency and the unspecific interference of siRNA on survivin expression 2 control transfections were performed, with 5 nM Alexa Fluor 488 labeled AllStars Negative control siRNA (Qiagen, Hilden, Germany) and RNAse free water, respectively. After elec- troporation, the cells were further cultured for 24h in 6-well plates in complete F15 medium Leibowitz, then cells were incu- bated with or without 50 uM etoposide for 24h at 37 ℃.
Apoptosis assay
The cells were stained for 30 min at room temperature with APC labeled Annexin V according to manufacturer’s instructions (BD Biosciences, San Jose, USA). After 2-min incubation with propid- ium iodide (PI), the cells were washed and the staining was assessed using a FACS Calibur system and Cell Quest™M Pro soft- ware (both BD Biosciences, San Jose, USA). As apoptotic cells were considered the cells that were both positive for Annexin V and PI. The rate of transfection was analyzed by the fluorescence intensity for the FITC channel (Alexa Fluor 488 labeled AllStars negative control siRNA cells vs. cells transfected with RNAse- free water) and by quantitative analysis of survivin RNA expres-
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a
1.0×1000
p<0.05
p<0.05
c
a
1.0×10-01
+b
1.0×10-02
1.0×10-03
1
c
2
S
3
1.0×10-04
1.0×10-05
1.0×10-06
1.0×10-07
Normal adrenal gland (n=12)
Adreno- cortical carcinoma (n=29)
Cortisol producing adenoma (n=8)
Aldostero n producing adenoma (n=8)
Endocrine inactive adenoma (n=8)
ACC cell lines (n=2)
4
5
6
b
3.5
p<0.0001
7
8
9
3.0
2.5
2.0
PCA low survival (n=17)
PCA high survival (n=16)
Principal component analysis (PCA) based ACC clusters
compared to no expression in the capsule (c-1, a) and very low expression (expression score 1) in the medulla (c-1, c). In malignant tissues the levels of cytoplasmic survivin varied between high (score 3) (c-4, magnification in c-5), intermediate (score 2) (c-9), low (score 1) (c-8) and no expression (score 0) (c-7) and was restricted to the tumor cells, the stromal cells lacking survivin expression. Negative control staining with unspecific rab- bit antibodies showed no staining, both in normal adrenal gland (c-3) and carcinoma tissue (c-6). Red-brown = Nova Red survivin specific staining; blue = hematoxylin staining of the nuclei. c-1, c-4: 100 x magnification; c-2, c-3, c-5 to c-9: 400 x magnification.
| Score | 0 | 1 | 2 | 3 |
|---|---|---|---|---|
| ACC total (n=154) | 5 (3%) | 22 (14%) | 70 (46%) | 57 (37%) |
| Primary tumor (n= 123) | 3 (2%) | 18 (15%) | 55 (45%) | 47 (38%) |
| Local recurrence (n=18) | 2 (11%) | 4 (22%) | 8 (45%) | 4 (22%) |
| Metastases (n=13) | 0 | 0 | 7 (54%) | 6 (46%) |
| Adrenal adenoma (n=15) (Cushing, Conn, Inactive) | 0 | 3 (20%) | 7 (47%) | 5 (33%) |
| Normal adrenal gland (n=4) | 0 | 2 (50%) | 2 (50%) | 0 |
Table 2 Survivin protein expres- sion in evaluable adrenocortical tissues.
sion in the siRNA transfected and untransfected cells. Three independent experiments were performed, each in triplicate and the results are expressed as mean value.
Circulating survivin specific cytotoxic T-cells (CTL) Peripheral blood mononuclear cells (PBMCs) from 29 ACC and 3 CPA patients with HLA-A2 haplotype, 6 healthy donors with HLA-A2 haplotype and 3 with HLA-A1 haplotype (· Table 1, Part C) were isolated using Ficoll® density centrifugation. From
each donor, 106 cells were incubated with APC labeled HLA-A2 restricted dextramer-peptide complexes specific for survivin (LMLGEFLKL), EBV (GLCTLVAML), and HIV (ILKEPVHGV), respec- tively, according to manufacturer’s instructions (Dako, Glosdrup, Denmark). The cytotoxic T-lymphocytes (CTL) were counter- stained with a FITC labeled mouse anti-CD8 antibody (clone DK25, Dako, Glosdrup, Denmark). As experimental control staining using APC labeled mouse isotype control instead of the dextramers was performed in all samples. The staining results
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were visualized using a FACS Calibur flow cytometer (BD Bio- sciences, San Jose, USA) and the analysis was performed with Cell Quest™M Pro software (BD Biosciences, San Jose, USA).
Statistical analyses
All statistical tests were done using the Prism 5.0 (GraphPad Software Inc., La Jolla, USA). Significance of differences between 2 groups were performed using the unpaired t-test and between 3 or more groups by ANOVA. A p-value of <0.05 was considered statistically significant with post-hoc analysis carried out by Kruskal-Wallis test and Dunn’s Multiple Comparison Test. All results are expressed as means +SD. Survival analyses were cal- culated using the Kaplan-Meier method and differences between groups were assessed with log-rank statistics. Overall survival (OS) was defined as the time from the date of the primary tumor excision to date of death or last analysis.
Results
▼
Survivin mRNA expression levels by real-time RT-PCR Survivin mRNA expression was significantly higher in ACC tissues and the 2 ACC cell lines than in normal adrenal glands and benign adrenocortical adenomas with mean 18S normal- ized CP values of 7.7x10-3+4x10-2 for ACC tissues and 4×10-4±1.2×10-4 for ACC cell lines vs. 1.3×10-6+9.7x10-7 for normal adrenal gland, 6.2×10-6+1.4x10-5 for CPA, 7.4×10-7± 3.2×10-7 for APA and 2.8x10-5+6x10-5 for EIA respectively (· Fig. 1a). However, within the group of 29 ACC samples sur- vivin mRNA expression was highly variable with 18S normalized CP values between 3.6×10-7 and 2.2 ×10-1 (· Fig. 1a).
Survivin mRNA expression levels by Expression Microarray Analysis (in silico) and correlation with survival
Using the expression array data from the Ann Arbor group [47], we found that survivin mRNA expression in the normal and benign tissues was comparable (normalized expression values 2.2±0.11 vs. 2.15±0.10, respectively, p>0.05) while it was sig-
a
c
100
100
HR=2.09 (0.9-4.85)
Overall survival (%)
p=0.08
75-
Overall survival (%)
75-
50-
50-
HR A vs. C =4.5 (1.6-13.3)
p=0.005
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Time (months)
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- survivin (2-3) n=98 -*- survivin (0-1) n=21
- A: survivin (2-3)/SF-1 (3) n=33 — B: survivin (2-3)/SF-1 (0-2) n=65 -+- C: survivin (0-1)/SF-1 (0-2) n=16
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HR A vs. C =7.3 (2.8-18.8)
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P<0.0001
HR A vs. C =4.5 (1.0-19.1)
p=0.04
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- A: survivin (2-3)/bcat (1) n=35
— B: survivin (2-3)/bcat (0) n=32
- A: survivin(2-3)/Glut-1(3) n=12 — B: survivin(2-3)/Glut1(0-2) n=84 --- C: survivin(0-1)/Glut-1(0-2) n=20
-*- C: survivin (0-1)/bcat (0) n=12
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nificantly higher in the ACC samples (2.39±0.16, p<0.05 com- pared with both, expression in normal adrenal and adrenocortical adenoma). Distribution of survivin mRNA expression values in the 33 ACC tissues according to principal component analysis (PCA) clusters showed a highly significant correlation between high survivin mRNA expression and shorter survival (2.89±0.05 vs. 2.58±0.04 for the tumors of patients in the PCA high survival cluster, p<0.0001) (~ Fig. 1b).
Survivin protein expression
From 154 evaluable ACC samples, only 3% of the samples were negative, 14% had a staining score of 1, 46% a score of 2 and 37% a score of 3 ( Fig. 1c and . Table 2). There was no significant difference between primary tumors and local recurrences, but all metastatic ACC samples had at least a moderate staining intensity. All of the 15 adrenocortical adenomas were survivin positive, 20% with a score of 1, 47% with a score of 2, and 33% with a score of 3. Survivin protein expression was also found in all the normal adrenal glands with equal distribution between scores 1 and 2 (@ Table 2). However, expression was restricted to the 3 cortical areas of the gland and the medulla ( Fig. 1b, c), whereas the capsular tissue ( Fig. 1a-c) remained negative.
Correlation of protein expression of survivin and other prognostic factors with clinical outcome
In 120 evaluable ACC patients, there was a strong trend associat- ing survivin expression with low survival rate. The median over- all survival in patients with low survivin expression (IHC score 0-1, n=21) was 95 months (95% CI: 19.4-171.5) vs. 30 months (14.6-44.8) in patients with high survivin expression (IHC score 2-3, n=98), p=0.053, HR=2.28 (0.99-5.28). This trend was con- firmed by a tumor stage adjusted analysis, p=0.086, HR=2.09 (0.90-4.85) (· Fig. 2a).
We further tried to improve the prognostic power by combining the information derived from the analyses with survivin with other factors already demonstrated to harbor prognostic poten- tial in ACC. When combined with beta-catenin expression, the median overall survival in patients with low expression of both, survivin and CTNNB1, (“low-low”) was 144 months (108.0- 180.5) compared to 31 months (8.4-54.3) in patients with high survivin and low CTNNB1 expression (“high-low”) and 30 months (8.1-52.2) for those with tumors expressing both pro- teins at high levels (“high-high”). The number of patients with low survivin and high beta catenin expression (“low-high”) was too small (n=2) to include in the statistical analysis. The hazard ratio (HR) for death for the patients with tumors with high expression of both survivin and beta catenin was 4.5 (1.0-19.1) compared to those with low expression of both proteins (p=0.04), independent of the tumor stage (· Fig. 2b). When we combined the results of survivin and SF-1 expression, the median survival of the “low-low” patients was 131.2 months (0-287.4) vs. 45 months (22.8-66.9) in the “high-low” and 16.3 months (4.9-27.7) in the “high-high” group. The hazard ratio for death was 4.5 (1.6-13.3) for the “high-high” patients compared to the “low-low” in a bivariate analysis including tumor stage (p=0.005) ( Fig. 2c). Finally, the data from the survivin analysis were combined with Glut-1 data. In this analysis, the median survival of patients with low expression of both factors was 94 months (59.2-129.0) vs. 81 months (60.8-102.4) for those with “high-low” expression and only 11.5 (8.2-14.7) months for those with “high-high” expression. The hazard ratio for death was 7.3 (2.8-18.8) for the patients with high-high expression
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Survivin mRNA expression (18s normalized)
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compared to those with both survivin and Glut-1 underex- pressed (p<0.0001) ( Fig. 2d).
Survivin functional analysis
To assess the extent to which survivin expression is involved in the anti-apoptotic mechanisms of ACC tumor cells, we knocked down expression by survivin siRNA transfection in the ACC cell- line SW-13.
The rate of transfection as measured by Alexa 488 labeled non- interfering siRNA fluorescence intensity was 92% and survivin siRNA expression knockdown as determined by real-time PCR was 93%, from a normalized CP value of 4.7x 10-4 to 3.3 x 10-5 ( Fig. 3a). The influence of survivin knockdown, with and without etoposide, was measured as rate of induced apoptosis by annexin V/propidium iodide (PI) staining (· Fig. 3b). Sur- vivin knockdown alone almost doubled the rate of apoptosis in the SW-13 cells (5-5.6 vs. 9.6%), reaching almost the same level
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| e | negative | positive |
|---|---|---|
| ACC | ||
| tumor bearing | 18 | 2 |
| tumor free | 8 | 1 |
| Controls | 9 | 0 |
Fig. 4 Presence of spontaneous survivin specific cytotoxic T-cells in the blood of ACC patients. PBMCs were isolated from the blood of 29 ACC and 6 healthy donors with HLA-A2 haplotype and 3 donors with HLA-A1 haplotype and stained with APC labeled HLA-A2 restricted dextramer- MHC I-peptide complexes specific for survivin b EBV c and HIV d and
counterstained with a FITC labeled antibody against CD8. Only the lymphocyte population was evaluated (a, gate R1). All control donors and 26 ACC patients (18 tumor-bearing and 8 tumor-free) did not have any survivin specific CTLs in circulation while 3 ACC patients (2 tumor-bearing and 1 tumor-free) had very low numbers of circulating survivin CTLs e.
as the untransfected cells treated with etoposide (9.6 vs. 11.3%). However, additional treatment with etoposide on the survivin siRNA transfected cells showed only a 23% increase in the apop- tosis rate (9.6 vs. 12.4%).
Circulating survivin specific cytotoxic T-lymphocytes (CTLs)
Finally, we investigated the presence of survivin-specific CTLs in the blood of patients with ACC. In 83% of all blood samples we could detect EBV peptide specific CTLs ranging from 4 to 196 cells per 100 000 PBMCs, but in none of the samples HIV peptide specific CTLs could be detected. However, only 3 blood samples could be identified that were positive for survivin CTLs, 1 belong- ing to tumor free and 2 to tumor bearing ACC patients. However, the frequency of reactive cells was quite low, between 4 and 30 per 100 000 cells ( Fig. 4).
Discussion
▼ In recent years, efforts have dramatically increased in identify- ing the main players involved in the mechanism of tumor resistance to chemotherapy as candidates for a successful immu- notherapy strategy [48-50]. As a member of the IAP family sur- vivin has attracted considerable attention and its overexpression has been associated with poor prognosis in numerous types of cancer [51]. This is the first study addressing the possible role of survivin in adrenocortical carcinoma.
In contrast to other studies that identify cytoplasmic survivin only in tumor and fetal tissue [1], we found survivin mRNA and protein expression both in neoplastic as in normal adrenocorti- cal tissues with high inter-individual variances. This concurs with the findings of Koch et al. in the adrenal gland medulla [52]. However, compared to their data, our real-time RT PCR analysis and our in silico analysis based on the microarray data already published [47] revealed a significantly higher survivin mRNA expression in the malignant tissues than in the benign and nor- mal adrenal gland. In addition, this analysis also revealed that
higher survivin expression correlates with poor prognosis. Although at mRNA level survivin expression was significantly higher in malignant than in benign/normal tissue, protein expression, as visualized by immunohistochemistry, showed a more even distribution between the 3 groups. Interestingly, sur- vivin protein expression was absent only in very few malignant tumors.
Patients with high intratumoral survivin protein expression had a twice as high mortality rate compared to those with absent or low survivin expression. The poor prognosis of survivin overex- pressing ACCs could be enhanced by combining the results with data from established prognostic markers. Of note, adding sur- vivin expression to the survival analyses improved the prognos- tic value of beta catenin [HR from 2.4 (1.3-4.6) [37] to 4.5 (1.0-19.1)], SF-1 [HR from 2.5 (1.3-4.6) [35] to 4.5 (1.6-13.3)], and glucose transporter 1 (Glut-1) [HR from 6.4 (3.4-12) [36] to 7.3 (2.8-18.8)]. These data suggest that survivin plays an impor- tant anti-apoptotic role in the molecular pathways of several molecules relevant for the ACC. A similar result was seen when we analyzed the smaller cohort with available mRNA expression results, with a median overall survival of 56 months in patients with high survivin expression compared to 96 months in the “low survivin” group [HR 1.70 (0.47-6.35)].
We also found evidence for a role of survivin in the apoptosis resistance of ACC in in vitro experiments, as its downregulation using siRNA was enough to double the spontaneous apoptosis rates. Especially in the case of ACC where efficient therapeutic options are scarce this could be helpful for the development of new therapeutic strategies. These include vaccination strategies to generate an antigen specific immune response against sur- vivin bearing tumor cells [53-55]. Furthermore, in recent years other methods have been developed that are based on inhibiting survivin expression or function instead of destroying survivin bearing tissue. These methods include the use of inhibitory RNA molecules [56-58], ribozymes [59-61] or antisense oligonucle- otides [62-64] targeting survivin and more recently the small molecule inhibitor of survivin function (YM155) [65,66], which is already tested in a phase II clinical trial [67,68]. Up to now, all
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these inhibitors showed good responses in vitro but only moder- ate responses in clinical trials. Thus, it has been suggested to combine them with conventional chemotherapy. However, as seen in the ACC cell line SW-13 inhibition of survivin does not always lead to a sensitization to chemotherapeutic drugs, probably due to the complexity of the pro and anti-apoptotic mechanisms with multiple players involved and redundant sig- naling pathways that vary from tumor to tumor.
In contrast to other tumors [69,70] we could not find a signifi- cant spontaneous cytotoxic T-cell immune response directed against survivin in our cohort. In only 10% of the studied patients a very low number of survivin specific CTL could be detected. This lack might be partly explained by the autoantigen nature of survivin probably combined with the fact that many ACCs secrete cortisol, a known immunogenic inhibitor of T-cell func- tion [71,72].
In conclusion, survivin is expressed in both normal and neoplas- tic adrenocortical tissue and there is evidence that high expres- sion of this protein is associated with poor clinical outcome. Furthermore, survivin seems to play an important role in the anti-apoptotic mechanisms of ACC that makes it a reasonable candidate for future therapeutic strategies.
Acknowledgements
▼
This work was supported by grants from the Deutsche Kreb- shilfe (Grant 107111 to M.F. and Grant 106 080 to B.A. and M.F.), the Deutsche Forschungsgemeinschaft Grant FA466/3-1 to M.F.), and the German Ministry of Research BMBF (Grant 01KG0501 to B.A. and M.F.).
Conflict of Interest
▼
The authors have no conflict of interest.
Affiliations
1 Department of Internal Medicine I, Endocrine and Diabetes Unit, University Hospital Würzburg, University of Würzburg, Würzburg, Germany
2 Department of Dermatology, University Hospital Würzburg, University of Würzburg, Würzburg, Germany
3 Department of General Dermatology, Medical University of Graz, Graz, Austria
4 Department of Pathology, University Hospital Tübingen, Tübingen, Germany
5 Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany
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