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Cancer Research 66, 10016-10023, October 15, 2006. doi: 10.1158/0008-5472.CAN-05-4079
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Loss of Caspase-8 Expression Does Not Correlate with MYCN Amplification, Aggressive Disease, or Prognosis in Neuroblastoma

Simone Fulda1, Christopher Poremba2, Bernd Berwanger3,4, Sabine Häcker1, Martin Eilers3, Holger Christiansen4, Barbara Hero5 and Klaus-Michael Debatin1

1 University Children's Hospital, Ulm, Germany; 2 Institute of Pathology, Heinrich-Heine-University, Düsseldorf, Germany; 3 Institute for Molecular Biology and Tumor Research; 4 University Children's Hospital, Marburg, Germany; and 5 Children's Hospital, Paediatric Oncology, University of Cologne, Cologne, Germany

Requests for reprints: Simone Fulda, University Children's Hospital, Eythstrasse 24, D-89075 Ulm, Germany. Phone: 49-731-5002-5980; Fax: 49-731-5002-6765; E-mail: simone.fulda{at}uniklinik-ulm.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inactivation of caspase-8 because of aberrant gene methylation has been associated with amplification of the MYCN oncogene and aggressive disease in neuroblastoma, suggesting that caspase-8 may function as tumor suppressor. However, the prognostic effect of caspase-8 in neuroblastoma has remained obscure. Therefore, we investigated caspase-8 expression and its correlation with established prognostic markers and survival outcome in a large cohort of neuroblastoma patients. Here, we report that loss of caspase-8 protein expression occurs in the majority (75%) of neuroblastoma and is not restricted to advanced disease stages. Surprisingly, no correlation was observed between caspase-8 expression and MYCN amplification. Similarly, ectopic expression of MYCN or antisense-mediated down-regulation of MYCN had no effect on caspase-8 expression in neuroblastoma cell lines. In addition, caspase-8 expression did not correlate with other variables of high-risk disease (e.g., 1p36 aberrations, disease stage, age at diagnosis, or tumor histology). Most importantly, loss of caspase-8 protein had no effect on event-free or overall survival in the overall study population or in distinct subgroups of patients. By revealing no correlation between caspase-8 expression and MYCN amplification or other established variables of aggressive disease, our findings in a large cohort of neuroblastoma patients show that inactivation of caspase-8 is not a characteristic feature of aggressive neuroblastoma. Thus, our study provides novel insight into the biology of this tumor, which may have important clinical implications. (Cancer Res 2006; 66(20): 10016-23)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neuroblastoma is the most common solid extracranial tumor of early childhood (1). Neuroblastoma patients with high-risk disease continue to exhibit a poor prognosis, even after intensive chemotherapy and autologous bone marrow transplantation (2, 3). Several tumor characteristics (e.g., amplification of the MYCN oncogene, loss of heterozygosity at chromosome 1p, or telomerase activity) are statistically significant indicators of poor prognosis for neuroblastoma patients (4–6). Although studies over the last years have uncovered several important molecular pathways involved in the pathogenesis or progression of neuroblastoma, the prognostic effect of many of these molecular factors has not yet been studied in detail.

Apoptosis, the intrinsic cell death program, plays a crucial role in the regulation of tissue homeostasis, and an imbalance between cell death and proliferation may result in tumor formation (7). Apoptosis pathways may be initiated through different entry sites, such as death receptors or mitochondria, resulting in activation of effector caspases (7). Stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily, such as CD95 (APO-1/Fas) or TNF-related apoptosis-inducing ligand (TRAIL) receptors, results in receptor aggregation and recruitment of Fas-associated death domain and caspase-8 to activated death receptors (8). On recruitment, caspase-8 becomes activated, which then cleaves downstream effector caspases (8). The mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis-inducing factor, Smac, or endonuclease G from the mitochondrial intermembrane space (9–11). Studies from caspase-8 knockout mice indicate that caspase-8 plays a necessary and nonredundant role in various forms of cell death (12). Interestingly, mutations in caspase-8 have only been identified at low frequency in some tumors (e.g., colorectal cancer or head and neck carcinoma; refs. 13, 14).

Apoptosis has been supposed to play a key role in neuroblastoma biology (15). For example, apoptosis may be involved in mediating spontaneous regression, one of the unique features of this tumor (16). By comparison, defects in apoptosis programs may contribute to tumor progression and resistance of neuroblastoma because killing of tumor cells by cytotoxic therapies is predominantly mediated through induction of apoptosis in target cells (15). Resistance to apoptosis can be acquired by cancer cells through a variety of means (e.g., loss of signaling molecules mediating cell death; ref. 17). In neuroblastoma, inactivation of caspase-8 by hypermethylation has become a hallmark of defective apoptosis in advanced disease, suggesting that caspase-8 may act as a tumor suppressor gene in neuroblastoma (18–21). To this end, the methylation status of caspase-8 has been linked to MYCN amplification in some studies (18, 22), but not in others (23, 24), and a direct effect of MYCN amplification on caspase-8 expression has not been described thus far. Based on these experimental findings, one would expect that caspase-8 expression bears prognostic effect for outcome in neuroblastoma, an issue that has not previously been addressed. We therefore investigated caspase-8 expression in a large cohort of well-characterized neuroblastoma patients treated according to the Cooperative German Neuroblastoma Trials, using cDNA microarray, tissue microarray, or Western blot analysis, and correlated caspase-8 expression with established markers of progressive disease and outcome.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Microarray
Patients. A total of 162 patients of the Cooperative German Neuroblastoma Trials NB90, NB95, and NB97 were analyzed by tissue microarray (Beecher Instruments, Sun Prairie, WI). Tumor samples were taken before chemotherapy. One hundred forty of 162 neuroblastoma tumor samples present on the tissue microarray were evaluable for quantification of caspase-8 expression by immunohistochemistry. The patient population comprised 55 of 140 (39.3%) stage I tumors, 25 of 140 (17.9%) stage II, 16 of 140 (11.4%) stage III, 38 of 140 (27.1%) stage IV, and 6 of 140 (4.3%) stage IVS tumors.

Immunohistochemistry. Three-micrometer sections were prepared from the tissue microarray and mounted onto silane-coated glass slides. Immunohistochemical analyses with mouse anti-caspase-8 (Upstate Biotechnology, Lake Placid, NY) were done using a standard LSAB protocol (ScyTek Laboratories, Logan, UT). Caspase-8 expression was evaluated by a pathologist, who was blinded to all clinical variables, semiquantitatively by percentage of stained tumor cells and staining intensity (negative, intermediate or strong caspase-8 staining in <1% of tumor cells; positive, intermediate or strong caspase-8 staining in >50% of tumor cells).

Analysis of chromosomal alterations and N-myc proto-oncogene (MYCN) amplification. Determination of the MYCN status was assessed in neuroblastoma tumors by fluorescence in situ hybridization (FISH), PCR, or Southern blot analysis. According to the European Network for Quality Assurance in Higher Education guidelines (25), MYCN amplification was defined as >4-fold increase of MYCN signal number in relation to the number of chromosome 2. Chromosomal aberrations in 1p36 were investigated in neuroblastoma tumors using FISH or PCR.

Statistical analysis of tissue microarray. To compare variables of interest, {chi}2 test, Fisher's exact test, or Mann-Whitney U test was used where appropriate. Survival curves were calculated according to Kaplan-Meier and compared with log-rank test. Event-free survival was calculated as the time from diagnosis to event or last examination if the patient had no event. Recurrence, progression of disease, and death from disease were counted as events. Overall survival was calculated as the time from diagnosis to death or last examination if the patient survived. Death resulting from therapy complications or from second malignancy was not counted as an event but censored for event-free survival and overall survival analysis.

Gene expression profiling. Expression profiles from 94 individual neuroblastoma tumor biopsies, which have previously been described, were obtained with a 4,608-cDNA human unigene chip (26). The tumors were selected to reflect distribution of tumor stages and MYCN amplification in the total tumor bank of 1,378 tumors distinct from those analyzed by tissue microarray (26). Statistical analysis of gene expression profiles was done as previously described (26). Briefly, to permit interspot and interarray comparisons, each signal was background corrected, and log 2–transformed red/green intensity ratios [spot intensity between tumor RNA (red) and SH-EP neuroblastoma cell line RNA (green)] were calculated and standardized. To compare the expression profile between two independent groups, the two-sample t statistic was used for every gene. Expression data are shown as box plots. The line in the middle represents the median of the expression. The whiskers show the upper and lower range of data, up to 1.5x the box length from the median. The box shows the range of data between the 25th and 75th percentiles. An outlier (more than 1.5x the box length from the median) is shown as a circle.

Cell cultures and chemicals. Neuroblastoma cell lines were maintained in RPMI 1640 or DMEM (Life Technologies, Inc., Eggenstein, Germany) as previously described (27). Tet-21/N neuroblastoma cells with tetracycline-controlled expression of MYCN were kindly provided by W. Lutz (Institute for Molecular Biology and Cancer Research, Marburg, Germany; ref. 28), SH-EP neuroblastoma cells stably transfected with control vector (SH-EP 007) or a vector containing a functional MYCN oncogene (WAC 2) by L. Schweigerer (University Children's Hospital, Göttingen, Germany; ref. 29), and NBL-S neuroblastoma cells transfected with vector control (NBV), MYCN sense (NBS), or MYCN antisense (two clones: NBAS4 and NBAS5) by S.L. Cohn (Northwestern University, Chicago, IL; refs. 30, 31). Chemicals were purchased from Sigma (Deisenhofen, Germany).

Tumor lysates. Sixty-five primary neuroblastoma specimens from children of the Cooperative German Neuroblastoma Trials, which have previously been described (ref. 26; Supplementary Table S1), were analyzed for caspase-8 protein expression by Western blot analysis. Cellular proteins were extracted from primary neuroblastoma tumor samples by lysing 100 mg of tumor tissue with 500 µL of ice-cold lysis buffer [150 mmol/L NaCl, 1% NP40, 40,5 mmol/L Tris buffer (pH 8), protease and phosphatase inhibitors]. Caspase-8 expression was quantified by densitometry using the NIH image software and classified as weak or absent (<20% of caspase-8 expression in SH-EP neuroblastoma cells) or moderate or strong (>20% of caspase-8 expression in SH-EP neuroblastoma cells) compared with caspase-8 protein expression in SH-EP neuroblastoma cells used as positive control.

Western blot analysis. Western blot analysis was done as previously described (27) using mouse anti-caspase-8 monoclonal antibody C15 (1:10 dilution of hybridoma supernatant; kindly provided by P.H. Krammer, German Cancer Research Center, Heidelberg, Germany), mouse anti-MYCN monoclonal antibody (1:1,000; BD Biosciences, Heidelberg, Germany), or ß-actin monoclonal antibody (1:10,000; Sigma) followed by goat anti-mouse immunoglobulin G (1:5,000; Santa Cruz Biotechnology). Enhanced chemiluminescence (Amersham Pharmacia, Freiburg, Germany) was used for detection. Expression of ß-actin was used to control for equal gel loading.

Reverse transcription-PCR. Reverse transcription-PCR (RT-PCR) for caspase-8 was done as previously described (27) using the following primer sequences (Interactiva Biotechnologie GmbH, Ulm, Germany):5'-CAGCATTAGGGACAGGAATC-3' and 5'-CAGTTATTCACAGTGGCCAT-3'. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as standard for RNA integrity and equal gel loading. PCR reaction products were run on a 1.5% agarose gel, stained with ethidium bromide, and visualized by UV illumination. Expression of GAPDH mRNA was used to control for integrity of mRNA and for equal gel loading.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Caspase-8 expression in neuroblastoma. To gain insight into the effect of caspase-8 in primary neuroblastoma, we determined caspase-8 protein expression in primary neuroblastoma tumor samples by immunohistochemistry on a tissue microarray chip containing 162 neuroblastoma samples, 140 of which were evaluable for quantification. Immunostaining of caspase-8 protein was weak or absent in 75% of primary neuroblastoma whereas 25% of neuroblastoma stained positive for caspase-8 protein (Fig. 1A and B , and data not shown). To extend these studies, we analyzed expression of caspase-8 protein by Western blotting in tumor lysates of a distinct group of 65 neuroblastoma tumor samples. Similarly, weak or absent expression of caspase-8 protein was detected in 77% of primary neuroblastoma (Fig. 1C; Supplementary Fig. S1). These data show for the first time in a large panel of primary neuroblastoma tumors that caspase-8 protein expression is low or absent in the majority of primary neuroblastoma.


Figure 1
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  		Figure 1. Expression of caspase-8 protein in primary neuroblastoma. A and B, analysis of caspase-8 protein expression in primary neuroblastoma by tissue microarray. Caspase-8 protein expression was analyzed by immunohistochemistry on a tissue microarray chip. Characteristic samples positive (A) or negative (B) for caspase-8 protein are shown. C, analysis of caspase-8 protein expression in primary neuroblastoma by Western blot. Caspase-8 and ß-actin protein expression in primary neuroblastoma tumor samples was analyzed by Western blot. Representative neuroblastoma tumor samples (#1-5) are shown. SH-EP neuroblastoma cells served as a positive control for caspase-8 protein expression (Co).

 
Caspase-8 expression and MYCN amplification. Methylation of gene regulatory sequences of caspase-8 has previously been associated with amplification of the MYCN oncogene in some, but not all, studies (18, 22–24). However, the question whether or not MYCN amplification has a direct effect on caspase-8 expression levels has not yet been addressed. We therefore correlated caspase-8 protein expression, assessed by tissue microarray, with MYCN amplification in primary neuroblastoma tumor samples. Surprisingly, we did not detect a significant association between caspase-8 immunostaining as determined by tissue microarray and MYCN amplification (Table 1A ; P = 0.8, {chi}2, Fisher's exact test). Moreover, we extended our studies to the group of 65 neuroblastoma tumor samples analyzed by Western blotting. Also in this group, caspase-8 protein levels did not correlate with MYCN amplification (P = 0.72, {chi}2, Fisher's exact test). Next, we obtained gene expression profiles from 94 individual tumor biopsies using a 4,608-cDNA human unigene chip, which has previously been described (26). Notably, gene expression profiling in these primary neuroblastoma revealed no statistically significant differential expression of caspase-8 mRNA in MYCN-amplified tumors when compared with tumors containing single-copy MYCN (Fig. 2 ; P = 1.0, two-sample t test).


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  		Table 1. Correlation of caspase-8 expression and variables of aggressive disease

 

Figure 2
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  		Figure 2. Caspase-8 expression and N-myc amplification. Gene expression profiles of caspase-8 were determined by cDNA microarray and are shown as box plots for MYCN-amplified (amp) and single-copy MYCN (non-amp) tumors. P = 1.00, two-sample t test.

 
Furthermore, to test whether or not caspase-8 expression is under direct control of the MYCN oncogene, we used three different models of neuroblastoma cell lines either with transfection-enforced overexpression of MYCN or with antisense-mediated down-regulation of MYCN. For inducible, ectopic expression of MYCN, we selected Tet-21/N cells, which are a derivative of SH-EP neuroblastoma cells with tetracycline-controlled expression of MYCN (28). Notably, ectopic expression of MYCN, induced on removal of tetracycline from the culture medium, did not alter expression levels of caspase-8 mRNA or protein in Tet-21/N neuroblastoma cells (Fig. 3A ). Moreover, we used a model consisting of the human neuroblastoma cell line SH-EP that had been stably transfected with either a control vector (SH-EP 007) or a vector containing a functional MYCN oncogene (WAC 2; ref. 29). Similar to inducible expression of MYCN, stable overexpression of MYCN had no effect on caspase-8 mRNA or protein levels (Fig. 3B). Furthermore, down-regulation of MYCN protein expression by an antisense approach in NBL-S neuroblastoma cells, which harbor increased MYCN protein levels in the absence of gene amplification (30, 31), had no effect on caspase-8 mRNA or protein expression (Fig. 3C). In addition, we extended our studies to a panel of neuroblastoma cell lines. Although low or absent caspase-8 mRNA or protein expression was frequently detected in neuroblastoma cell lines harboring MYCN amplification, cell lines without MYCN amplification also lacked caspase-8 (SH-SY5Y, SK-N-SH, and SK-I-N) and cell lines in which caspase-8 was detected despite MYCN amplification (NB1 and NGP) were also identified (Fig. 3D; Supplementary Table S2). Together, these data obtained in neuroblastoma cell lines and primary neuroblastoma tumor samples imply that caspase-8 expression is not a consequence of increased MYCN expression in neuroblastoma.


Figure 3
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  		Figure 3. Effect of MYCN on caspase-8 expression in neuroblastoma cell lines. A and B, effect of MYCN overexpression on caspase-8 expression. Expression of caspase-8 or GAPDH mRNA or caspase-8, MYCN, or ß-actin protein was assessed by RT-PCR or Western blot analysis in Tet-21/N neuroblastoma cells with tetracycline-controlled expression of MYCN (A) or in SH-EP neuroblastoma cells stably transfected with a functional MYCN oncogene (WAC 2) or vector control (SH-EP 007; B). C, effect of down-regulation of MYCN on caspase-8 expression. Expression of caspase-8 or GAPDH mRNA or caspase-8, MYCN, or ß-actin protein was assessed by RT-PCR or Western blot analysis in NBL-S neuroblastoma cells transfected with vector control (NBV), MYCN sense (NBS), or MYCN antisense (NBAS4 and NBAS5). D, caspase-8 expression in neuroblastoma cell lines. Expression of caspase-8 or GAPDH mRNA or caspase-8, MYCN, or ß-actin protein was assessed in neuroblastoma cell lines by RT-PCR or Western blot analysis.

 
Caspase-8 expression and advanced disease. Because inactivation of caspase-8 has previously been implied as a characteristic feature of high-risk neuroblastoma (32), we next asked whether or not caspase-8 expression was associated with adverse prognostic variables. To address this question, we correlated caspase-8 expression with various established variables used for prognostic stratification of neuroblastoma. Gene expression profiling in primary neuroblastoma tumors revealed no statistically significant differential expression of caspase-8 mRNA in stage I versus stage IV disease, irrespective of whether both MYCN-amplified and nonamplified tumors or only single-copy MYCN tumors were analyzed (Fig. 4 ; P = 1.0, two-sample t test). Similarly, we found no stage-specific expression of caspase-8 protein in primary neuroblastoma when caspase-8 protein expression was determined by tissue microarray (Table 1B; P = 0.7, {chi}2 test). Furthermore, caspase-8 protein expression, as assessed by tissue microarray analysis, did not correlate with other prognostic variables such as 1p36 aberrations (Table 1C; P = 1.0, {chi}2 test), age at diagnosis (Table 1D; P = 0.29, Mann-Whitney U test), or tumor histology [e.g., histologic grading according to Hughes (Table 1E; P = 0.7, {chi}2 test) or Shimada classification (Table 1F; P = 1.0, {chi}2 test)]. Thus, expression of caspase-8 did not correlate with any of the tested variables of advanced disease.


Figure 4
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  		Figure 4. Caspase-8 expression and disease stage. Gene expression profiles of caspase-8 were determined by cDNA microarray and are shown as box plots for stage I versus stage IV in both MYCN-amplified and MYCN-nonamplified tumors (A) or for single-copy MYCN tumors (B). P = 1.00, two-sample t test.

 
Caspase-8 expression and survival. We then went on to evaluate the prognostic value of caspase-8 protein expression in neuroblastoma, as assessed by tissue microarray. All patients were treated according to the protocols of the Cooperative German Neuroblastoma Trials (NB90, NB95, and NB97). Importantly, expression of caspase-8 protein did not predict event-free or overall survival in Kaplan-Meier analysis for the total group of 140 neuroblastoma patients. The cumulative survival curves for event-free survival of patients with negative or positive caspase-8 immunostaining for this total group of neuroblastoma patients are shown in Fig. 5A . In addition, we assessed whether caspase-8 expression has a prognostic effect in distinct subsets of neuroblastoma patients. Interestingly, Kaplan-Meier analysis also revealed no significant differences in event-free or overall survival when localized tumors, stage IV disease, MYCN-amplified or nonamplified tumors, or localized tumors without MYCN amplification were analyzed (Fig. 5B-F, and data not shown). We also extended these studies to the cohort of 65 neuroblastomas, which were analyzed for caspase-8 protein expression by Western blotting. Also in this group of neuroblastomas, expression of caspase-8 protein had no effect on event-free or overall survival in Kaplan-Meier analysis (data not shown; P = 0.36 for event-free survival and P = 0.38 for overall survival, log-rank test).


Figure 5
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  		Figure 5. Caspase-8 expression and survival. The Kaplan-Meier curves show the probability of event-free survival (EFS) in terms of the level of caspase-8 protein expression (solid line, caspase-8 positive; broken line, caspase-8 negative) determined by tissue microarray for all stages (A; P = 0.36), localized disease (B; P = 0.13), stage IV disease (C; P = 0.51), MYCN nonamplified tumors (D; P = 0.49), MYCN amplified tumors (E; P = 0.99), or localized tumors without MYCN amplification (F; P = 0.26). The survival curves were analyzed by the log-rank test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inactivation of caspase-8 by hypermethylation has been suggested to be a hallmark of defective apoptosis and aggressive disease in neuroblastoma, suggesting that caspase-8 may act as a tumor suppressor gene (32). Despite these assumptions, the prognostic effect of caspase-8 expression in neuroblastoma has remained obscure. Therefore, we investigated caspase-8 expression and its correlation with established prognostic markers and survival outcome in a large cohort of neuroblastoma patients.

Importantly, we found loss of expression of caspase-8 protein in the majority of neuroblastoma (75%), irrespective of disease stage. In contrast, the only one previous study on caspase-8 protein expression in primary neuroblastoma reported loss of the caspase-8 protein in only 40% (4 of 10 tumors), which may, however, be due to the low sample number of this study (33), whereas most studies in the past focused on mRNA expression of caspase-8 in neuroblastoma (18, 22, 23). Surprisingly, in contrast to previous studies using cell lines or a limited number of patient samples, our study revealed a lack of correlation between caspase-8 expression and MYCN amplification in primary neuroblastoma samples. The notion that loss of caspase-8 expression is not a characteristic for aggressive neuroblastoma is further supported by our findings that caspase-8 expression did not correlate with various other variables of high-risk disease (e.g., 1p36 aberrations, tumor stage, age at diagnosis, or tumor histology). Most importantly, our study shows in a large population of well-characterized neuroblastoma patients that caspase-8 protein expression levels had no effect on event-free or overall survival. This conclusion holds true for the entire patient population or distinct subgroups of patients (e.g., neuroblastoma patients with metastatic disease or with MYCN amplification).

Thus, our findings show that inactivation of caspase-8 is not a characteristic feature of aggressive neuroblastoma harboring MYCN amplification as previously supposed by some reports (18, 22). Notably, these studies focused on association between MYCN amplification and the methylation status of the caspase-8 gene rather than caspase-8 expression (18, 22). The preferential epigenetic inactivation of caspase-8 in neuroectodermal tumors with MYC overexpression, including neuroblastoma, medulloblastoma, or neuroendocrine lung tumors, was taken as further evidence that proteins of the Myc family may negatively regulate caspase-8 (34–36). However, the issue of whether or not caspase-8 methylation is associated with MYCN amplification in neuroblastoma has also controversially been discussed (23, 24). Whereas caspase-8 methylation has almost exclusively been found in neuroblastoma patient samples or tumor cell lines with MYCN gene amplification in some studies (18, 22), no such correlation was detected by other investigators (23, 24). In contrast to these earlier reports on MYCN amplification and caspase-8 methylation, we tested for an association of MYCN amplification and expression of caspase-8. By examining distinct patient populations by cDNA microarray, tissue microarray, or Western blot analysis, we found no correlation between MYCN amplification and caspase-8 mRNA or protein expression. In addition, our studies in neuroblastoma cell line models clearly show that MYCN has no direct effect on caspase-8 expression, in line with a recent study (37). Because we detected no association between MYCN amplification and expression of caspase-8, we did not investigate whether MYCN amplification would affect the methylation status of caspase-8. Thus, in light of the lack of correlation between caspase-8 expression and various variables of aggressive disease including MYCN amplification, the previous view of MYCN-driven inactivation of caspase-8 as a hallmark of high-risk neuroblastoma may need to be revised. Our data support an alternative model, where loss of caspase-8 is not directly regulated by MYCN and may occur in the ontogeny of neuroblastoma development even before MYCN amplification. Because absence of caspase-8 has recently been reported in neural stem cells (38), lack of caspase-8 expression may merely reflect the developmental status of the neuroblast at the time of malignant transformation.

The lack of prognostic effect of caspase-8 expression in neuroblastoma may also be explained by redundant strategies for escaping apoptosis, which determine the resistance of this tumor to treatment (15). For example, down-regulation of TRAIL or CD95 receptors or enhanced expression of antiapoptotic proteins, such as survivin or Bcl-2, has been described in neuroblastoma (15). Thus, signal transduction to cell death may be blocked by multiple mechanisms in neuroblastoma cells, which may, at least in part, compensate for each other.

In contrast to neuroblastoma, loss of caspase-8 protein expression has recently been reported to occur only in the minority of primary medulloblastoma (16%), where it significantly correlated with unfavorable survival outcome (39). This points to distinct roles of caspase-8 in tumor formation or tumor progression in different neuroectodermal tumors. Because apoptosis exerts an essential function in neuroectodermal development, inactivation of apoptosis pathways (e.g., by loss of caspase-8 expression) may be especially important for the pathogenesis of neuroectodermal tumors. In line with this notion, down-regulation of caspase-8 has primarily been observed in a variety of neuroectodermal tumors (34, 35, 39).

Despite the lack of prognostic significance of caspase-8 expression in Kaplan-Meier survival analysis, our data, however, do not exclude that caspase-8 levels may have an effect on treatment response. End points such as event-free or overall survival are complex and can be influenced by various biological properties including rate of tumor growth, susceptibility to cell death stimuli, metastatic potential, angiogenic factors, as well as accessibility to treatment approaches. Thus, these end points do not directly measure response to therapy. The notion that caspase-8 expression may have an effect on response to chemotherapy or irradiation is supported by findings that neuroblastoma cells lacking caspase-8 regain sensitivity to anticancer drugs on restoration of caspase-8 expression (19, 20, 40). In addition, caspase-8 expression may influence tumor control by the immune system because neuroblastoma cells lacking caspase-8 have been shown to be more resistant to death receptor triggering via the CD95 or the TRAIL system (19, 20). Accordingly, caspase-8 levels may set the threshold for apoptosis sensitivity in response to chemotherapy or tumor attack by the immune system. Therefore, restoration of caspase-8 expression by targeted therapeutics may sensitize neuroblastoma cells for cytotoxic therapies, thereby enhancing treatment response. Thus, a prospective study is planned to directly correlate caspase-8 expression with patients' treatment response in neuroblastoma (e.g., clearance of tumor cells from the bone marrow after induction chemotherapy).

By showing that loss of caspase-8 expression occurs in the majority of neuroblastoma and is not restricted to advanced stages of the disease as previously assumed, our study provides novel insight into the biology of this tumor, which may have important clinical implications.


   Acknowledgments
 
Grant support: Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe, the Bundesministerium für Forschung und Technologie, the Ministry of Science, Research and Arts of Baden-Württemberg, Das Interdisziplinäre Zentrum für Klinische Forschung Ulm, Wilhelm-Sander-Stiftung, Else-Kröner-Stiftung, the Deutsche Kinderkrebsstiftung, and the European community (K-M. Debatin and S. Fulda).

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.

We thank P.H. Krammer for anti-caspase-8 antibody, W. Lutz, L. Schweigerer and S.L. Cohn for providing cell lines, and P. Miller and M. Preissinger for expert technical assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 11/14/05. Revised 6/27/06. Accepted 7/20/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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