In this study, we have investigated the participation of caspase-10 in TRAIL resistance of NB cells and described the specific role of individual caspase-10 isoforms in DR-mediated apoptosis in various human NB and adult tumour cell lines. We revealed a co-silencing of caspase-8 and -10 protein expression in N-type cells, a sub-type of apoptosis-resistant NB cells. In contrast, TRAIL-sensitive S-type cells were shown to express both caspases. However, reduced levels of caspase-10 were detected in these cells as compared with normal primary cells (adrenal gland and PBL). Decreased caspase-10 expression was also shown in adult tumour cell lines, supporting previously reported decreased caspase-10 expression in transformed T- and B-cell lines.4
Moreover, we show that the absolute level of endogenous caspase-10 mRNA expression is significantly weaker than the level of caspase-8 mRNA in all cell types investigated, which suggests an independently controlled function for caspase-10.
ShRNA-mediated caspase-8 knock-down experiments in SH-EP cells clearly indicated that the low endogenous caspase-10 expression level in NB cells was not sufficient to initiate an apoptotic cascade, even in the presence of a weak amount of residual caspase-8. Cell sensitivity to DR stimulation was shown to be directly dependent on initiator caspases expression levels. Indeed, TRAIL sensitivity of IGR-N-91 cells restored for caspase-8 expression was directly dependent on caspase-8 expression levels.27
Similarly, FasL sensitivity of caspase-8-deficient Jurkat I9-2 cells was shown to correlate with endogenous caspase-10 content.5
Moreover, the induction of caspase-8 or -10 expression in NB cells using either demethylating agent or IFN-γ
resulted in enhanced cellular sensitivity to death ligands.25, 28, 29, 30
Altogether, these findings indicate that not only caspase-8, but also caspase-10 levels need to be tightly controlled, and that caspase-10 downregulation is likely to significantly influence and modulate NB cells sensitivity to DR-induced apoptosis.
In addition to the demonstration of a co-silencing of initiator caspases related to aggressive and apoptosis-resistant NB tumours cells, our data identified a reduced level of both initiator caspases expression in adult tumour cells. This feature, which was linked to a correspondingly decreased sensitivity to DR-mediated cell death, is likely to represent a general mechanism of resistance to cell death developed by tumour cells.
Caspase-10 silencing was previously reported to occur by post-transcriptional control in adult and paediatric tumour cells.15, 19
Our data, in contrast, indicate that caspase-10 silencing in NB cells essentially occurs at the transcriptional level, as the relatively low amounts of caspase-10 mRNA measured by real-time PCR directly correlated with protein expression measured by immunoblotting in N- and S-type cells. This is compliant with studies showing that caspase-10 mRNA expression in TRAIL-resistant NB cells could be restored by the methyltransferase inhibitor 5-ADC.28
To address more specifically the function of caspase-10 in TRAIL or FasL-mediated cell death in tumour cells, we have explored the role of individual caspase-10 isoforms. Stable caspase-10A or -10D overexpression in caspase-8-positive SH-EP, SK-N-AS, SW480 and BJAB cells displayed a strong pro-apoptotic activity on TRAIL stimulation. Although caspase-10A or -10D only sensitise SW480 and BJAB cells to Fas stimulation, NB cells remained resistant because of their minimal Fas and high FLIP expression levels.25, 31
In addition, stable overexpression of caspase-10A or -10D isoforms in caspase-8/-10-silenced NB cells resulted in restoration of TRAIL sensitivity, indicating that, when expressed at sufficient levels, caspase-10A and -D isoforms are fully functional and can substitute for caspase-8 in TRAIL-mediated apoptosis in NB cells, as reported for Jurkat I9-2 cells.15
Our results also show that caspase-8 and -10 can initiate apoptosis independently of each other in NB cells.
When stably overexpressed, caspase-10B and -10G isoforms, in contrast, were either non-functional or even displayed anti-apoptotic activity. Caspase-10G resembles caspase-8L in lacking the active proteolytic domain, but retains the two DEDs. Our results suggest that, depending on the cellular context, caspase-10G may be either inactive (SK-N-AS, SW480) or act as a dominant-negative inhibitor of apoptosis (SH-EP, BJAB). In this case, caspase-10G is likely to interfere, like caspase-8L or viral FLIP proteins, with the recruitment of endogenous initiator caspases to activated receptors.32, 33
The differential pro- or anti-apoptotic activity of caspase-10A and -D and caspase-10B and -G, respectively, was observed in all tumour cell lines analysed, indicating that the opposing properties of caspase-10 isoforms are not restricted to NB. Nevertheless, caspase-10B was previously shown to have a pro-apoptotic activity in MCF-7 overexpressing exogenous caspase-3 (ref. 34
Interestingly, the inactive or anti-apoptotic caspase-10B isoform is highly homologous to the pro-apoptotic caspase-10D isoform, except for the last 49 amino acids that generate a caspase-10B-specific p12 fragment. The generation of a chimeric protein caspase-10A/B allowed us to demonstrate that the differential activity of caspase-10B resulted from its unique C
-terminal end, which also mediated its destabilisation via the proteasome pathway. When exogenously expressed, caspase-10B protein level was always weaker than that of other caspase-10 isoforms. This was most likely the result of its weaker stability, as caspase-10B mRNA expression level was higher than that of caspase-10A, -10D or -10G mRNAs (Supplementary Information). Interestingly, analysis of the caspase-10B amino acid sequence (using Conserved Domains search service from NCBI) revealed that several amino acids involved in the protease dimer interface are lacking in caspase-10B p12 subunit, as compared with caspase-10A, -10D or -8. As dimerisation of apical caspases is crucial for their activation,35
the formation of less stable dimers could result in a reduced ability of caspase-10B to be auto-activated or to a weaker proteolytic activity. Indeed, caspase-10A and -D were very quickly activated on TRAIL exposure, while caspase-10B activation needed increased amounts of TRAIL. Interestingly, caspase-10B exhibited a short half-live, since its basal expression level could be enhanced by the proteasome inhibitor Lactacystin in SH-EP and SW480 cells, in contrast to other caspase-10 isoforms or caspase-8. Interestingly the steady-state levels of other DISC components, such as Trail-R2 and FLIP, have also been shown to be modulated by the proteasome pathway via ubiquitination.36, 37
In conclusion, our results obtained with a panel of NB and adult tumour cell lines uncovered an important (critical) role for caspase-10 in the initiation of DR apoptosis. A differential pro- or anti-apoptotic role for distinct caspase-10 isoforms was revealed. Moreover, our data revealed the transcriptional silencing of caspase-10 in tumour, such as NB as an essential mechanism of resistance to DR signalling. In tumour as in normal cells, the balance in the relative expression of initiator caspases-8 and -10 isoforms may thus provide an additional keen (subtle) regulatory mechanism of tumour sensitisation to apoptotic stimuli. Identification of therapeutic strategies capable to differentially modulate the expression of particular caspase-10 isoforms would help in the further sensitisation of highly apoptosis-resistant tumours such as NB.