Identification of a novel pro-apoptotic role of NF-κB in the regulation of TRAIL- and CD95-mediated apoptosis of glioblastoma cells

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Abstract

We recently reported that nuclear factor-kappa B (NF-κB) promotes DNA damage-triggered apoptosis in glioblastoma, the most common brain tumor. In the present study, we investigated the role of NF-κB in death receptor-mediated apoptosis. Here, we identify a novel pro-apopotic function of NF-κB in TRAIL- and CD95-induced apoptosis. Inhibition of NF-κB by overexpression of the dominant-negative IκBα-superrepressor (IκBα-SR) significantly decreases tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)- or CD95-induced apoptosis. Vice versa, activation of NF-κB via overexpression of constitutively active IκB kinase complex (IKK)β (IKK-EE) significantly increases TRAIL-mediated apoptosis. Intriguingly, NF-κB inhibition reduces the recruitment of Fas-associated death domain and caspase-8 and formation of the death-inducing signaling complex (DISC) upon stimulation of TRAIL receptors or CD95. This results in reduced TRAIL-mediated activation of caspases, loss of mitochondrial potential and cytochrome c release in IκBα-SR-expressing cells. In comparison, NF-κB inhibition strongly enhances TNF-α-mediated apoptosis. Comparative studies revealed that TNF-α rapidly stimulates transcriptional activation and upregulation of anti-apoptotic proteins, whereas TRAIL causes apoptosis before transcriptional activation. Thus, this study demonstrates for the first time that NF-κB exerts a pro-apoptotic role in TRAIL- and CD95-induced apoptosis in glioblastoma cells by facilitating DISC formation.

Main

Glioblastoma is the most aggressive brain tumor, bearing a very poor outcome (DeAngelis, 2001). The failure of conventional therapy is mainly due to an aberrant regulation of multiple signaling pathways, leading to diffuse infiltration, genomic instability and resistance to apoptosis (Furnari et al., 2007). Apoptosis can be initiated via two pathways, the extrinsic (death receptor) and the intrinsic (mitochondrial) pathway (Fulda and Debatin, 2006). Binding of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or CD95 ligand to their cognate receptors leads to formation of the death-inducing signaling complex (DISC) via the recruitment of the adapter protein Fas-associated death domain (FADD) and caspase-8, causing activation of caspase-8 and direct cleavage of downstream effector caspases (Fulda and Debatin, 2006). The extrinsic pathway can be linked to the intrinsic pathway via caspase-8-mediated cleavage of Bid, which translocates to the mitochondria, resulting in mitochondrial outer membrane permeabilization and an amplification of the apoptotic signal (Fulda and Debatin, 2006). TRAIL is considered as a promising agent for anticancer treatment, as it induces apoptosis in various cancers including glioblastoma (Humphreys and Halpern, 2008). In addition to apoptosis, TRAIL is implicated in the activation of several other pathways such as the NF-κB cascade, thereby modulating the apoptotic signal (Humphreys and Halpern, 2008).

The transcription factor NF-κB is a dimer composed of proteins belonging to the NF-κB/Rel family (Karin et al., 2002). In resting cells, NF-κB is sequestered in the cytoplasm by an inhibitory IκB protein, predominantly IκBα (Karin et al., 2002). Upon stimulation, the IκB kinase complex (IKK) becomes activated, leading to proteasomal degradation of IκBα and the translocation of NF-κB to the nucleus (Karin et al., 2002). Generally, NF-κB is considered as anti-apoptotic, for example, in TNF-α signaling, as it is implicated in the transcriptional activation of proteins promoting cell survival (Karin et al., 2002). In line with this notion, several publications suggest NF-κB to be responsible for mediating resistance to TRAIL-induced apoptosis (Plantivaux et al., 2009). More recently, NF-κB has also been described to promote apoptosis in a cell type- and stimulus-dependent manner (Radhakrishnan and Kamalakaran, 2006).

We previously reported that inhibition of NF-κB decreases anticancer drug-induced apoptosis in glioblastoma cells, demonstrating a pro-apoptotic role of NF-κB in DNA damage-triggered apoptosis in glioblastoma (Karl et al., 2009). In the present study, we investigated the role of NF-κB in death receptor-induced apoptosis.

NF-κB inhibition decreases death receptor-induced apoptosis

To elucidate the role of NF-κB in death receptor-mediated apoptosis of glioblastoma, we generated T98G, A172 and U87MG glioblastoma cell lines that stably express the dominant-negative superrepressor mutant IκBα-S(32, 36)A (IκBα-SR), which cannot be phosphorylated and degraded (Karl et al., 2009). Control experiments showed that expression of IκBα-SR efficiently inhibited TRAIL-induced degradation of IκBα and NF-κB DNA binding in glioblastoma cells (Supplementary Figure 1). Interestingly, inhibition of NF-κB significantly decreased TRAIL-induced apoptosis, dose-dependently in all three glioblastoma cell lines (Figure 1a). Similarly, NF-κB inhibition profoundly reduced apoptosis upon treatment with TRAIL-R2-specific antibodies as well as CD95-triggered apoptosis (data not shown, Figure 1b). In sharp contrast, apoptosis was significantly enhanced upon TNF-α stimulation (Figure 1a), in line with the well-established anti-apoptotic function of NF-κB in TNF-α signaling (Doi et al., 1999). To investigate whether the differential role of NF-κB in TRAIL versus TNF-α signaling results from activation of different NF-κB subunits, we performed supershift analysis. This revealed that both TRAIL and TNF-α similarly triggered a DNA binding complex consisting of the NF-κB subunits p65/p50 and p50/p50 (Supplementary Figure 2a). Furthermore, TNF-α rapidly enhances NF-κB DNA binding, resulting in a rapid and strong increase in NF-κB transcriptional activity (Supplementary Figures 2b and c), while TRAIL triggered apoptosis before NF-κB transcriptional activation (Supplementary Figures 2c and d). In comparison, the kinetics of TNF-α-induced apoptosis upon NF-κB inhibition was relatively slow (Supplementary Figure 2d). This set of experiments demonstrates for the first time that NF-κB exerts a pro-apoptotic role in TRAIL- or CD95-induced apoptosis in glioblastoma cells compared with the anti-apoptotic function of NF-κB in the TNF-α pathway.

Figure 1
figure1

Inhibition of NF-κB decreases TRAIL- and CD95-antibody-induced apoptosis. T98G, A172 and U87MG glioblastoma cells were obtained from ATCC (Manassas, VA, USA), stably transduced with empty vector control (white bars) or IκBα-S(32, 36)A (black bars) as previously described (Karl et al., 2009) and treated for 48 h with TRAIL (R&D Systems, Wiesbaden, Germany) at the indicated concentrations or 30 ng/ml TNF-α (Biochrom, Berlin, Germany) (a) or anti-CD95 antibody (b) (Trauth et al., 1989). Apoptosis was assessed by fluorescence-activated cell sorting analysis of DNA fragmentation of propidium iodide-stained nuclei as previously described (Karl et al., 2009). Data are shown as mean±s.e.m of three independent experiments performed in triplicate (a: middle panel, b: all panels) or duplicate (a: left and right panels). Statistical significance was assessed by Student's t-test (two-tailed distribution, two-sample, unequal variance). *P<0.05; **<0.001.

NF-κB inhibition reduces TRAIL-induced activation of caspases and mitochondrial outer membrane permeabilization

To gain insight into the underlying molecular mechanisms responsible for the pro-apoptotic function of NF-κB in TRAIL- or CD95-mediated apoptosis, we analyzed the different steps in the death receptor signaling cascade. Monitoring of caspase activation revealed that NF-κB inhibition delayed and reduced activation of caspase-8, -3, -2 and -9, as well as processing of Bid to tBid (Figure 2a). The broad-range caspase-inhibitor zVAD.fmk completely blocked TRAIL-induced apoptosis, demonstrating caspase dependency (Figure 2b). Moreover, loss of mitochondrial membrane potential and release of cytochrome c were substantially decreased upon inhibition of NF-κB (Figures 2c and d).

Figure 2
figure2

NF-κB promotes TRAIL-induced activation of caspases and mitochondrial outer membrane permeabilization. (a) Cells were treated with 5 ng/ml TRAIL (T98G) or 25 ng/ml TRAIL (U87MG) for the indicated times and activation of caspases was assessed by western blotting as previously described (Karl et al., 2009; Mader et al., 2010) using the following antibodies: rabbit anti-Bid (Cell Signaling, Beverly, MA, USA), mouse anti-caspase-2 (BD Bioscience, Heidelberg, Germany), rabbit anti-caspase-3 (Cell Signaling), mouse anti-caspase-8 (Alexis, San Diego, CA, USA) and rabbit anti-caspase-9 (BD Bioscience). β-Actin (Sigma, Deisenhofen, Germany) served as loading control. (b) Glioblastoma cells transduced with empty vector control (white bars) or with a vector containing IκBα-SR (black bars) were treated for 48 h with 5 ng/ml TRAIL (T98G) or 25 ng/ml TRAIL (U87MG) in the presence or absence of 25-μmol zVAD.fmk (Bachem, Heidelberg, Germany). Apoptosis was assessed by fluorescence-activated cell sorting (FACS) analysis of DNA fragmentation of propidium iodide-stained nuclei. (c, d) Glioblastoma cells expressing empty vector control (white bars) or IκBα-SR (black bars) were treated for the indicated times with 5 ng/ml TRAIL (T98G) or 25 ng/ml TRAIL (U87MG). Mitochondrial transmembrane potential (c) and cytochrome c release (d) were assessed by FACS analysis as described previously (Mader et al., 2010). In a, c and d, data are shown as mean±s.e.m of three independent experiments performed in triplicate. Statistical significance was assessed by Student's t-test. *P<0.05; **<0.001.

Other studies have shown that NF-κB can function in a pro-apoptotic manner by regulating the ratio between death and decoy receptors (Farhana et al., 2005; Kang et al., 2010). However, we observed no NF-κB-dependent alterations in the cell surface expression of TRAIL receptors or CD95 during stimulation with TRAIL, while the decrease in TRAIL-R2 expression upon treatment with TRAIL occurred in the presence and absence of NF-κB (Supplementary Figure 3a). Reduced TRAIL-R2 expression on the surface may be due to TRAIL-mediated internalization of TRAIL-R2 (Kohlhaas et al., 2007), as downregulation of TRAIL-R2 was inhibited when the experiment was performed at 4 °C (Supplementary Figure 3a). Together, these results indicate that NF-κB modulates TRAIL-stimulated signaling and apoptosis upstream of caspase activation and loss of mitochondrial outer membrane permeabilization.

Next, we explored whether NF-κB alters apoptosis sensitivity by modulating the expression of endogenous death receptor ligands or pro- or anti-apoptotic proteins as described in previous studies (Shou et al., 2002; Campbell et al., 2004; Farhana et al., 2005; Poppelmann et al., 2005). Although TRAIL mRNA levels were slightly reduced upon stimulation with TRAIL in IκBα-SR overexpressing cells, we observed no concomitant changes in TRAIL protein expression (Supplementary Figure 3b). In addition, CD95L mRNA as well as CD95L protein expression was not affected upon inhibition of NF-κB or exposure to TRAIL (Supplementary Figure 3b). These findings indicate that NF-κB-mediated changes in TRAIL or CD95 ligand are probably not responsible for the pro-apoptotic function of NF-κB in TRAIL-induced apoptosis. Similarly, NF-κB inhibition did not alter expression levels of cFLIP proteins (FLIPL, FLIPS), Bcl-2 family members (Bcl-2, Bax and Bcl-XL) or X-linked inhibitor of apoptosis protein in the presence or absence of TRAIL (Supplementary Figure 3c). Interestingly, NF-κB inhibition profoundly suppressed constitutive expression of cIAP2, which has been implicated in NF-κB p65-mediated resistance to TNF-α-induced apoptosis in glioblastoma cells (Zhao et al., 2011). In addition, NF-κB inhibition prevented the TRAIL- or TNF-α-stimulated upregulation of cIAP2 and cIAP1, as well as the increase in FLIPL and FLIPS upon TNF-α stimulation (Supplementary Figure 3c). Of note, the increase in the NF-κB target genes cIAP1, cIAP2, FLIPL and FLIPS was more pronounced upon stimulation with TNF-α compared with TRAIL (Supplementary Figure 3c), in line with our findings that TNF-α is more potent than TRAIL to trigger NF-κB transcriptional activity (Supplementary Figure 2c).

NF-κB inhibition reduces TRAIL- or CD95-triggered DISC formation

We then examined the key apical event in death receptor-mediated apoptosis, that is, DISC formation upon stimulation with TRAIL. Intriguingly, we found that inhibition of NF-κB substantially reduced the recruitment of FADD, caspase-8 and its cleavage fragments and the p43 cleavage product of cFLIPL to stimulated TRAIL receptors, while these proteins were equally expressed in cell lysates (Figure 3). Similarly, analysis of the CD95 DISC revealed that less FADD and active caspase-8 were recruited to the CD95 DISC in cells expressing IκBα-SR (Supplementary Figure 4a). This demonstrates that NF-κB inhibition interferes with TRAIL- or CD95-triggered DISC formation, resulting in reduced recruitment of FADD and caspase-8 to activated TRAIL receptors or CD95, consistent with reduced TRAIL- or CD95-induced apoptosis upon NF-κB inhibition. In comparison, NF-κB inhibition prevented the TNF-α-stimulated recruitment of cIAP2 to TNFR1, while it did not affect the recruitment of other TNFR1-interacting proteins such as cIAP1, TRADD and TRAF2 (Supplementary Figure 4b). FADD and caspase-8 were not detected in the TNFR1 membrane-associated complex I, in line with a previous report (Micheau and Tschopp, 2003).

Figure 3
figure3

Blocking of NF-κB decreases TRAIL DISC formation. T98G, A172 and U87MG cells containing empty vector control or IκBα-SR were stimulated for 30 min with 1-μg/ml flag-tagged TRAIL. DISC analysis was performed by immunoprecipitation and western blotting using 1 μg/ml Flag-tagged TRAIL (Alexis, Grünberg, Germany) and the following antibodies: mouse anti-caspase-8 (Alexis), mouse anti-FADD (BD Bioscience), mouse anti-FLIP (Alexis) and rabbit anti-TRAIL-R2 (Chemicon, Billerica, MA, USA). After lysis using a buffer containing 50 mM Tris–HCl, 1% (v/v) Triton-X 100, 150 mM NaCl, protease inhibitor cocktail (Roche, Mannheim, Germany), 1 μg/ml Flag-tagged TRAIL was also added to the untreated samples. The TRAIL receptor-associated DISC was immunoprecipitated from lysates using 1.25 μg/ml mouse-anti Flag M2 antibody (Sigma). Elution of the precipitate was done by adding 10 μl pan-mouse IgG Dynabeads (Invitrogen, Karlsruhe, Germany) and by overnight rotation. Samples were washed three times with washing buffer I (50 mM Tris–HCl, 500 mM NaCl, 1% (v/v) Igepal CA-630 (NP-40)), and once with washing buffer II (25 mM Tris–HCl) and analyzed by western blotting. *IgG heavy chain, **IgG light chain.

We next analyzed TRAIL binding to TRAIL-R2 to determine whether differences in DISC formation are due to altered affinity of TRAIL to its receptor upon NF-κB inhibition. However, we found no differences in the binding of TRAIL to TRAIL-R2 in IκBα-SR overexpressing compared with control cells (Supplementary Figure 4c), indicating that NF-κB does not alter the binding of TRAIL to its receptors. Furthermore, we examined translocation of TRAIL receptors into lipid rafts, which may regulate DISC formation and activity (Pennarun et al., 2010). We observed decreased recruitment of TRAIL-R2 into lipid rafts in IκBα-SR overexpressing cells compared with vector control cells (Supplementary Figure 5), indicating that NF-κB inhibition decreases TRAIL-R2 redistribution into lipid rafts.

NF-κB activation by constitutively active IKK-β enhances TRAIL-induced apoptosis and DISC formation

To verify the pro-apoptotic function of NF-κB in a second cellular system, we ectopically activated NF-κB by overexpression of the gain-of-function mutant IKK-β-S(177 181)E (IKKβ-EE) (Figure 4a). Expression of constitutively active IKK-β resulted in constitutive NF-κB activation as demonstrated by an increased binding of NF-κB to the DNA and increased NF-κB transcriptional activity (Figure 4b). Indeed, NF-κB activity enhanced the recruitment of FADD and caspase-8 to the TRAIL DISC upon stimulation with TRAIL (Figure 4c). As a consequence, TRAIL-induced apoptosis was significantly increased in cells expressing constitutively active IKKβ (Figure 4d). These data confirm by an independent approach that NF-κB promotes TRAIL-induced DISC formation and apoptosis.

Figure 4
figure4

Effect of enhanced NF-κB activity on TRAIL-induced apoptosis. T98G glioblastoma cells were generated by transiently transfecting Phoenix producer cells (Orbigen, San Diego, CA, USA) either with empty pCFG5-IEGZ vector or pCFG5-IEGZ vector containing IKKβ-S(177 181)E using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendation and subsequent retroviral spin transduction of T98G cells and selection with Zeozin (Invivogen, San Diego, CA, USA). In a, ectopic expression of wildtype and mutant IKK-EE was assessed by western blotting using rabbit anti-IKKα/β (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In b, DNA binding activity of NF-κB after stimulation with 10 ng/ml TNF-α for 1 h was determined by electrophoretic mobility shift assay as previously described (Karl et al., 2009) using the sequence of specific oligomer for NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (left panel). NF-κB transcriptional activity was assessed by transiently transfecting cells using Fugene-HD (Roche Applied Science, Mannheim, Germany) with constructs containing Firefly and Renilla genes and determining acitvities of Firefly and Renilla luciferase after stimulation with 5 ng/ml TRAIL or 10 ng/ml TNF-α for 24 h in the presence of 25 μmol zVAD.fmk as described previously (Karl et al., 2009) (right panel). Values are depicted as fold increase of luciferase activity relative to unstimulated control out of two independent experiments performed in triplicate with mean±s.e.m. (c) T98G cells containing empty vector control or IKKβ-EE (EE) were stimulated for 30 min with 1-μg/ml flag-tagged TRAIL. DISC analysis was performed by immunoprecipitation and western blotting as described in legends for Figure 3. *IgG heavy chain, **IgG light chain. (d) T98G cells stably expressing empty vector control (white bars) or IKKβ-EE (black bars) were treated for 24 h with TRAIL at indicated concentrations or 30 ng/ml TNF-α, and apoptosis was assessed by fluorescence-activated cell sorting analysis of DNA fragmentation of propidium iodide-stained nuclei. Values are shown as are mean±s.e.m of three independent experiments performed in triplicate. *P<0.05; **<0.001.

Finally, we used mouse embryonic fibroblasts from NEMO deleted mice, in which TRAIL- or TNF-α-stimulated NF-κB activation was inhibited (Supplementary Figures 6a and b). In line with a previous study (Ravi et al., 2001), mouse embryonic fibroblasts from NEMO knockout mice were significantly more sensitive to treatment with TRAIL or TNF-α compared with wildtype mouse embryonic fibroblasts (Supplementary Figure 6c), indicating that NF-κB exerts its pro-apoptotic function in a cell type-dependent manner.

In this study, we show for the first time that NF-κB exerts a pro-apoptotic function in TRAIL- or CD95-induced apoptosis. Inhibition of NF-κB by overexpression of the dominant-negative IκBα-superrepressor (IκBα-SR) significantly decreases TRAIL- or CD95-induced apoptosis. Vice versa, stimulation of NF-κB due to overexpression of constitutively active IKKβ significantly increases TRAIL- or CD95-mediated apoptosis. Intriguingly, we found that NF-κB promotes the formation of the TRAIL or CD95 DISC by enhancing the recruitment of FADD and caspase-8 to the activated TRAIL receptors or CD95. While the NF-κB-mediated alterations in death receptor signaling at this early event might point to constitutive changes of NF-κB target genes that prime glioblastoma cells for TRAIL receptor- and CD95-induced apoptosis, we did not detect alterations in the expression pattern of key apoptosis regulatory proteins upon modulation of NF-κB activity that could explain this pro-apoptotic function of NF-κB.

In contrast to this pro-apoptotic role of NF-κB in TRAIL- or CD95-induced apoptosis, NF-κB inhibition enhances TNF-α-mediated apoptosis, consistent with the well-established anti-apoptotic function of NF-κB in the TNF-α signaling pathway. This differential regulation of death receptor-triggered apoptosis is associated with rapid transcriptional activation of NF-κB and upregulation of anti-apoptotic proteins upon TNF-α stimulation, whereas TRAIL causes apoptosis before NF-κB transcriptional activation. By comparison, TRAIL and TNF-α trigger similar NF-κB DNA binding complexes.

To date, nothing has been reported about a possible link between NF-κB and DISC formation. Previously, cross-talks between death receptor and survival pathways such as protein kinase C and ERK1/2 signaling pathways are described to modulate DISC formation (Pennarun et al., 2010). Furthermore, several posttranslational modifications of DISC components, such as death receptor O-glycosylation, S-palmitoylation or S-nitrosylation of TRAIL-R1 or phosphorylation of caspase-8 or cFLIP are described to either promote or hamper DISC formation (Pennarun et al., 2010). In addition, DISC formation has been shown to be regulated by aggregation of death receptors stimulated by ceramide release (Pennarun et al., 2010). Our findings indicate that differential lipid raft translocation of TRAIL receptors may contribute to reduced TRAIL sensitivity upon NF-κB inhibition, while TRAIL binding to TRAIL-R2 or TRAIL receptor internalization upon ligand binding are not affected by NF-κB activity. As far as DNA damage-triggered apoptosis is concerned, we previously reported that NF-κB increases DNA strand breaks and apoptosis upon treatment with anticancer drugs in glioblastoma (Karl et al., 2009). It is important to note that NF-κB exerts this pro-apoptotic function in a cell type-dependent manner, as NF-κB inhibition enhances TRAIL-induced apoptosis in mouse embryonic fibroblasts from NEMO knockout mice (Supplementary Figure 6) or in neuroblastoma cells (Ammann et al., 2009). A pro-apoptotic role of NF-κB has also been described in several models of neuronal cell death, for example, in response to ischemia, glutamate or N-methyl-D-aspartate receptor triggering (Mattson, 2005).

In conclusion, we identify a novel pro-apoptotic function of NF-κB in the context of TRAIL- or CD95-mediated cell death in glioblastoma cells. Thus, this study provides novel insight into the regulation of apoptosis by NF-κB and points to its putative tumor suppressor function in glioblastoma. These findings have important implications for the design of strategies to bypass apoptosis resistance of glioblastoma.

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Acknowledgements

We thank R Marienfeld (Ulm, Germany) for providing NEMO knockout mouse embryonic fibroblasts. This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, European Community (ApopTrain, APO-SYS) and IAP6/18 (to SF), and by a scholarship of the International Graduate School of Molecular Medicine Ulm University (to CJ).

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Correspondence to S Fulda.

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Jennewein, C., Karl, S., Baumann, B. et al. Identification of a novel pro-apoptotic role of NF-κB in the regulation of TRAIL- and CD95-mediated apoptosis of glioblastoma cells. Oncogene 31, 1468–1474 (2012) doi:10.1038/onc.2011.333

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Keywords

  • TRAIL
  • apoptosis
  • NF-κB
  • glioblastoma
  • CD95

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