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The Journal of Immunology, 2007, 178: 1923-1930.
Copyright © 2007 by The American Association of Immunologists, Inc.

Selective Inhibition of I{kappa}B Kinase Sensitizes Mantle Cell Lymphoma B Cells to TRAIL by Decreasing Cellular FLIP Level1

Gaël Roué, Patricia Pérez-Galán, Mónica López-Guerra, Neus Villamor, Elias Campo and Dolors Colomer2

Hematopathology Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In an attempt to circumvent the intrinsic resistance of mantle cell lymphoma (MCL) cells to apoptosis, we have analyzed their sensitivity to the extrinsic apoptotic signal triggered by TRAIL. We show here that TRAIL can trigger apoptosis in a majority of MCL cell lines and primary cultures, irrespective of receptor levels, Bcl-2 family members, or caspase regulator expression. MCL sensitivity to TRAIL was closely linked to the activity of the NF-{kappa}B p50 factor and to the consequent expression of cellular FLIP (c-FLIP), which accumulated into the TRAIL-dependent complex in resistant cells. c-FLIP transient knockdown overcame MCL resistance to TRAIL, while NF-{kappa}B inhibitors differentially modulated TRAIL cytotoxicity. Indeed, bortezomib increased TRAIL cytotoxic effects in sensitive cells, but led to the intracellular accumulation of c-FLIP, impeding full synergistic interaction. In contrast, the I{kappa}B kinase inhibitor BMS-345541 led to decreased c-FLIP expression and allowed all MCL samples to undergo TRAIL-mediated apoptosis. These results present the combination of TRAIL stimulation and I{kappa}B kinase inhibition as a new approach to MCL therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mantle cell lymphoma (MCL)3 is a B lymphoid neoplasm with a mature phenotype, genetically characterized by the t(11;14)(q13;q32) translocation leading to cyclin D1 overexpression with the consequent deregulation of cell cycle at the G1-S checkpoint. MCL’s clinical behavior is aggressive, with an average overall survival of 3 years. MCL patients have a short survival rate and few are cured with current therapies (1). Recently, promising results have been reported in relapsed or refractory MCL treated with the proteasome inhibitor bortezomib (2). Our group has identified the proapoptotic protein Noxa as a crucial mediator of bortezomib-mediated cell death and the consequent activation of intrinsic mitochondrial apoptosis pathway in MCL cells, irrespective of p53 status (3).

Specific targeting of malignant cells while preserving the normal counterpart remains a crucial point for clinical cancer treatments. In this field, TRAIL (Apo2L), initially identified by its high sequence homology to other TNF family members (4), can selectively induce apoptosis in cancer cells (5, 6). TRAIL kills by binding one of the two cell surface receptors, either death receptor 4 (DR4/APO2/TRAIL-R1) or death receptor 5 (DR5/KILLER/TRAIL-R2/TRICK2) (7). Once activated, these transmembrane receptors trimerize and assemble a death-inducing signaling complex (DISC) constituted by their death domains, the Fas-associated death domain (FADD) adaptor protein, and the inactive proenzymatic form of the apoptosis-initiating proteases caspase-8 or -10. These caspases self-activate by proteolysis and in turn activate the effector caspase-3, either by direct processing (type I cells), or through cleavage of the proapoptotic Bcl-2 family member Bid, and engagement of mitochondrial apoptotic pathway, which involves release of cytochrome c, formation of the apoptosome and activation of caspase-9 (type II cells). TRAIL also associates with the decoy inhibitory receptors DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4), whose expression was initially thought to determine cellular sensitivity to TRAIL, and to allow normal surrounding cells to escape from TRAIL-mediated apoptosis (8). Interestingly, TRAIL receptor-mediated apoptosis has been reported to be inhibited by cellular FLIP (c-FLIP), through suppression of either recruitment of procaspase-8 by FADD or autocatalytic activation of caspase-8 (9). c-FLIP can be present in short (c-FLIPS) and long (c-FLIPL) forms, known to depend on NF-{kappa}B activation in some models (10).

NF-{kappa}B is a ubiquitously expressed family of transcription factors that includes p65 (RelA), p50, p52, c-Rel, and RelB, all of which can bind to DNA and form heterodimers or homodimers, the p65/p50 dimer being the predominant transcription factor complex. In most cell types, this complex exists in the cytoplasm bound to a family of inhibitors of {kappa}B (I{kappa}B). Following cellular stimulation by specific inducers, I{kappa}B is phosphorylated by the I{kappa}B kinase (IKK) complex and then degraded by the 26S proteasome. Subsequently, NF-{kappa}B translocates to the nucleus, where it regulates the transcription of several genes implicated in both survival and apoptotic signaling pathways (11), such as c-FLIP, antiapoptotic members of the Bcl-2 family of proteins (Bcl-2, Bcl-xL) and members of the inhibitor of apoptosis protein (IAPs) family (c-IAP1/2, X-chromosome linked IAP (XIAP), survivin) (10, 12). Inappropriate activation of the NF-{kappa}B pathway has been shown to contribute to tumor formation while increased DNA binding of NF-{kappa}B is associated with tumor resistance to chemotherapy (13, 14).

In this context, prompted by the specific antitumor activity of TRAIL in several models, we analyzed the sensitivity of MCL primary tumor cells and cell lines to TRAIL-induced apoptosis and addressed the shortcomings in the cell death signaling by pharmacological inhibition of the NF-{kappa}B-dependent pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines

HBL-2, UPN-1, Granta-519, Jeko, Rec-1, and JVM-2 cell lines were used. Genetic characterization of these cell lines has been published recently (15). JVM-2 and Rec-1 cells have been previously shown to harbor a genomic loss at 8p21 (16), presumably affecting the TRAIL receptor locus. MCL cell lines (0.3–0.5 x 106 cells/ml) were cultured in RPMI 1640 culture medium (Invitrogen Life Technologies), supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies), 2 mM glutamine, 50 µg/ml penicillin-streptomycin, and 100 µg/ml of the antimycoplasm agent normocin (Amaxa). Granta-519 cells were cultured with DMEM culture medium (Invitrogen Life Technologies) instead of RPMI 1640 medium.

Isolation and culture of primary cells

Tumor cells from peripheral blood or spleen from 10 MCL patients diagnosed according to the World Health Organization classification (17) who had not received treatment for the previous 3 mo were studied (Table I). Immunophenotype was performed by immunohistochemistry on tissue sections and/or by flow cytometry on cell suspensions. PBLs from MCL patients and three healthy donors were isolated by Ficoll/Hypaque sedimentation (Seromed). Tumor cells from spleen were obtained after squirting with RPMI 1640 culture medium using a fine needle. An informed consent was obtained from all cases in accordance with the Ethical Committee of the Hospital Clinic (Barcelona, Spain). Cells were either used directly or cryopreserved in liquid nitrogen in the presence of 10% DMSO and 20% FCS. Manipulation due to freezing/thawing did not influence cell response. Mononuclear cells were cultured in X-vivo 10 medium (BioWhittaker) at a density of 1–2 x 106 cells/ml. All cultures were maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide.


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  		Table I. Characteristics of MCL patients

 
Cytofluorimetric assessment of apoptosis

Cells were cultured as indicated with recombinant human TRAIL (Alexis Biochemicals), bortezomib (PS-341/Velcade; Millennium), BAY-117082 (Calbiochem) or 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline (BMS-345541; Bristol-Myers Squibb), and cell viability was measured with an Annexin VFITC conjugate combined with 0.5 µg/ml propidium iodide (Bender Medsystems). To measure mitochondrial membrane potential ({Delta}{psi}m), cells were incubated at 37°C for 15 min in the presence of 20 nM 3,3'-diexyloxacarbocyanine iodide (DiOC6[3]; Molecular Probes) and immediately analyzed for fluorochrome incorporation in a FACScan (BD Biosciences).

Western blotting

Total protein extracts were obtained from 5 x 106 cells lysed by a 30-min incubation in a buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, and 1 mM sodium orthovanadate). Fifty micrograms of solubilized proteins were separated on a 12% SDS-PAGE and transferred onto an Immobilon P membrane (Millipore). For protein immunodetection, membranes were blocked in TBS-Tween 20 containing 5% nonfat milk, and incubated with specific primary Abs, followed by HRP-labeled anti-mouse (Sigma-Aldrich) or anti-rabbit (DakoCytomation) secondary Ab. Ab binding was detected using an ECL detection system (Amersham) coupled to a LAS3000 Fujifilm device. Equal protein loading was confirmed with {alpha}-tubulin or beta-actin detection.

Antibodies

Abs against the following proteins were used: Mcl-1, Bak, and FADD (Santa Cruz Biotechnology), Bcl-2 (DakoCytomation), Bcl-xL, Bax, and caspase-3 (BD Pharmingen), Bim, and I{kappa}B{alpha} (Calbiochem), caspase-9 (New England Biolabs), XIAP, Bid, and phospho-I{kappa}B{alpha} (Cell Signaling), survivin (Abcam), c-FLIP (Alexis), caspase-8 and {alpha}-tubulin (Oncogene Research), beta-actin (Sigma-Aldrich), and PARP (Roche).

Detection of intracellular and membrane proteins

A total of 3 x 105 cells were fixed for 15 min with 4% paraformaldehyde in PBS and permeabilized for 10 min in 0.1% saponin. Cells were then stained for 30 min with a specific Ab against the active form of caspase-3 (BD Pharmingen), followed by staining with an anti-rabbit FITC-labeled secondary Ab (Sigma-Aldrich). TRAIL receptors membrane expression was measured by staining cells with PE-labeled mAbs directed against the ectodomain of DR4, DR5, DcR1, and DcR2 receptors (Abcam). Briefly, 5 x 105 cells were washed in PBS, incubated for 30 min with 10 µl of each Ab, and then washed twice in PBS. For the analysis of these receptors on normal B cells, whole PBLs from healthy donors were costained for 30 min with 5 µl of anti-CD3-FITC and anti-CD19-PerCP Abs (BD Biosciences), to identify B cells by flow cytometry as the CD3–/CD19+ population. A total of 10,000 stained cells was then analyzed by flow cytometry.

RNA isolation and real-time RT-PCR

Total RNA was isolated using the guanidinium thiocyanate method (TRIzol; Invitrogen Life Technologies) and 1 µg of RNA was retrotranscribed to cDNA using the cDNA archive kit (Applied Biosystems). mRNA expression was analyzed in duplicate by quantitative RT-PCR on the ABI Prism 7700 sequence detection system using predesigned Assay-on-demand primes and probes (Applied Biosystems). mRNA expression was analyzed by the comparative cycle threshold (Ct) method ({Delta}{Delta}Ct). beta-glucuronidase was used as an internal control and mRNA expression levels were given as arbitrary units.

DISC analysis

DISC precipitation was performed using biotin-tagged rTRAIL (Bio-TRAIL) (18), provided by Dr. A. López-Rivas (Centro Andaluz de Biología Molecular y Medicine Regeneravita, Sevilla, Spain). 75 x 106 MCL cells were incubated for 1 h with Bio-TRAIL, washed three times in ice-cold PBS, and lysed in 3 ml of lysis buffer (30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, fully completed with protease inhibitors as above) for 30 min on ice followed by centrifugation at 15,000 x g for 10 min at 4°C. To provide an unstimulated receptor control, Bio-TRAIL was added to lysates from untreated cells. The TRAIL DISC was then precipitated using 30 µl of streptavidin-agarose beads at 4°C overnight. Precipitates were washed six times with lysis buffer, and receptor complexes were eluted with 30 µl of sample buffer. Western blotting was performed as described above.

NF-{kappa}B factor activity assay

Nuclear extracts were prepared from MCL cells using the BD Transfactor Extraction kit (BD Biosciences). A total of 20 x 106 cells were washed once in cold PBS and lysed by a 15-min incubation in 5 volumes of cell lysis buffer supplemented with 1 µM DTT and protease inhibitors. Cells were disrupted by using a narrow-gauge needle and centrifuged for 20 min at 10,000 x g. Nuclear pellets were resuspended in complete nuclear extraction buffer, passed through a narrow-gauge needle, and centrifuged for 5 min at 20,000 x g. Five micrograms of nuclear extracts were incubated into a BD Chemiluminescent Transfactor kit plate in the presence of polyclonal Abs directed against either p50, p52, p65, RelB, or c-Rel, followed by incubation with a HRP-conjugated anti-rabbit Ab. A chemiluminescence signal was acquired as above, and quantification was performed with Image Gauge software version 4.0 (Fujifilm).

RNA interference assay

For transient down-regulation assays, JVM-2 cells were electroporated in the Nucleofector system (Amaxa) with an small-interfering RNA (siRNA) double-stranded oligonucleotide targeting the exon 6 common to both human c-FLIPL and c-FLIPS isoforms (Ambion). The sense and antisense strains were 5'-GGAUCCUUCAAAUAACUUC-3' and 5'-GAAGUUAUUUGAAGGAUCC-3', respectively. As a negative control, we used an irrelevant nonsilencing siRNA (5'-UUCUCCGAACGUGUCACGU-3'). Briefly, 7 x 106 JVM-2 cells were resuspended in 100 µl of R cell Nucleofector solution containing 0.5 or 5 µM of double-stranded siRNAs and electroporated with the A23 Nucleofector program. Cells were transferred to culture plates and cultivated at 3 x 106 cells/ml for 3 h. Then, dead cells were removed by low-speed centrifugation and viable cells were cultured at 1 x 106 cells/ml for 3 h before analysis of c-FLIP expression and TRAIL stimulation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TRAIL induces apoptosis in MCL cell lines independent of TRAIL receptor levels

We assessed the cytotoxic effect of human recombinant TRAIL on six human MCL cell lines, which differ in their p53-dependent pathway status, growth characteristics, and sensitivity to cytotoxic drugs (3, 15). Cells were incubated using TRAIL doses ranging from 1 to 1 000 ng/ml and cytotoxicity was measured by annexin V labeling of externalized phosphatidylserine residues. We observed three different patterns of sensitivity to TRAIL among these cell lines (Fig. 1A). The highly sensitive group included the UPN-1, Jeko, and HBL-2 cell lines, with TRAIL LD50 of 2.3 ± 0.2 ng/ml, 7.0 ± 1.6 ng/ml, and 8.0 ± 1.4 ng/ml, respectively. In the intermediate group, including Rec-1 (LD50 of 21.5 ± 1.9 ng/ml) and Granta-519 (LD50 of 30.0 ± 0.8 ng/ml), the cytotoxic effect of TRAIL did not significantly increase with doses higher than 50 ng/ml. Finally, JVM-2 was the only cell line resistant to TRAIL even when doses up to 1 000 ng/ml were used. Accordingly, Jeko and Granta-519 cells underwent a rapid time-dependent mitochondrial depolarization affecting ~55% of the cells 16 h after TRAIL addition, whereas the {Delta}{psi}m remained unaltered in JVM-2 cells incubated with TRAIL (Fig. 1B).


Figure 1
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  		FIGURE 1. Activation of TRAIL cell death pathway in MCL cell lines. A, Cytofluorimetric analysis of annexin V after a 24 h treatment with increasing concentrations of human recombinant TRAIL in MCL cells. B, Detection of {Delta}{psi}m loss in three representative MCL cell lines treated for up to 24 h with TRAIL at 10 ng/ml (Jeko) or 100 ng/ml (Granta-519, JVM-2). C, Flow cytometry analysis of membrane expression of the four TRAIL receptors (dark line) in MCL cell lines and normal B lymphocytes. Shaded areas correspond to unlabeled cells. *, Rec-1 and JVM-2 are the only cell lines showing a deletion of the 8p21.3 locus by comparative genomic hybridization and multiplex-fluorescence in situ hybridization analysis.

 
To assess whether sensitivity to TRAIL was related to the expression of its receptors in MCL cells, we analyzed the membrane expression of DR4, DR5, DcR1, and DcR2 by flow cytometry. Fig. 1C shows that all MCL cell lines expressed the four receptors, regardless of the presence of a genomic loss at 8p21.3, the region where the genes coding for the DR4 and DR5 receptors are located. DR4 and DR5 were highly expressed compared with normal B lymphocytes, DR5 being the predominant receptor in all the samples analyzed. The decoy inhibitory receptors DcR1 and DcR2 were poorly expressed in both normal and MCL cells. Using normal PBLs as calibrator, high levels of DR4 and DR5 mRNA were detected in all but UPN-1 cell lines, Granta-519, JVM-2, and HBL-2 being the cell lines with the highest DR4 and DR5 mRNAs levels (data not shown), contrasting with the membrane expression and pointing to a possible posttranscriptional regulation of these genes in MCL cells, as previously described in melanoma cells (19). These results revealed a lack of correlation between the basal expression of TRAIL receptors and the response to TRAIL in MCL cell lines, because JVM-2 and Granta-519 cells presented the highest levels of DR4 and DR5 receptors and the lowest sensitivities to TRAIL.

c-FLIP is a mediator of MCL cell sensitivity to TRAIL

We subsequently analyzed apoptosis regulators whose activity may have an impact on TRAIL-driven apoptotic pathway in MCL cells. Western blot analysis of the Bcl-2 family of proteins showed a heterogeneous expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, and a highly variable expression of the proapoptotic BH3-only Bim among all the MCL cell lines (Fig. 2A). HBL-2 and Granta-519 cells showed high levels of Bcl-2 that might be due to an amplification of the 18p21 region involving the BCL-2 gene (15). However, Bcl-2 was not expressed in UPN-1 cells. The heterogenous expression of the proapoptotic gene BIM in MCL cell lines may be due to either amplification or deletion affecting the 2q13 region in these cells (20). No differences in the expression of the proapoptotic proteins Bax and Bak were observed. The caspases -9 and -3, the IAP proteins, and the caspase activator Smac/DIABLO, were expressed in all MCL cell lines (Fig. 2A). Analysis of the proteins that compose the DISC complex revealed a notable accumulation of the DISC inhibitor c-FLIP in the less sensitive and resistant cell lines Rec-1, Granta-519, and JVM-2 (Fig. 2A), whereas other cell lines presented low levels of this inhibitor. Furthermore, the analysis of c-FLIP mRNA levels showed a good correlation between c-FLIP gene transcription and protein expression, JVM-2 being the cell line with the highest expression at both levels (Fig. 2B). DISC precipitation further confirmed that after TRAIL treatment c-FLIP accumulated in the TRAIL-dependent DISC preferentially in the resistant cell line, impeding caspase-8 processing into the p43/44 fragments (Fig. 2C). Thus, among the main intracellular regulators of cell death, c-FLIP appeared to be the main factor whose expression and activity correlated with the response to TRAIL.


Figure 2
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  		FIGURE 2. Expression of the main apoptosis regulators and evaluation of c-FLIP activity in MCL cell lines. A, Western blot analysis of the Bcl-2 family proteins (upper panel), caspases and caspases regulators (middle panel), and DISC components (lower panel) in the six MCL cell lines ordered according to their respective TRAIL LD50. B, Real-time RT-PCR quantification of c-FLIP mRNA levels in MCL cell lines. Results are given in arbitrary units, using JVM-2 cells as calibrator. C, c-FLIP recruitment into the TRAIL-dependent DISC in MCL cells. Jeko and JVM-2 cells were incubated for 1 h with Bio-TRAIL (500 ng/ml). TRAIL receptor complexes were precipitated and presence of caspase-8, FADD, and c-FLIP was analyzed by Western blotting. *, An unspecific band due to streptavidin-agarose. D, JVM-2 cells were electroporated in the presence of a nonsilencing (ns) or c-FLIP siRNA, and 6 h after transfection cells were examined for c-FLIP protein level and, E, for sensitivity to TRAIL (100 ng/ml, 16 h).

 
To further characterize the role of c-FLIP in TRAIL-mediated apoptosis in MCL cells, we used an siRNA approach to selectively down-regulate c-FLIP protein synthesis. A specific oligonucleotide that targets both short and long forms of c-FLIP RNA allowed a dose-dependent decrease in c-FLIP expression in JVM-2 cells, when compared with cells transfected with an irrelevant nonsilencing siRNA (Fig. 2D). This decrease allowed the JVM-2 cells transfected with the c-FLIP siRNA to efficiently undergo TRAIL-mediated apoptosis after incubation with 100 ng/ml TRAIL for 24 h (Fig. 2E). Similar results were observed when Granta-519 cells were transfected in the same conditions (data not shown). These results confirmed the inhibitory function of c-FLIP in determining MCL sensitivity to TRAIL.

Effects of NF-{kappa}B pathway inhibition on c-FLIP expression and sensitivity to TRAIL

Because c-FLIP synthesis has been shown to be regulated at a transcriptional level by the NF-{kappa}B family of transcription factors (10), we further analyzed the basal DNA-binding activity of the five NF-{kappa}B proteins, p50, p52, p65, c-Rel, and RelB, in nuclear protein extracts from the six MCL cell lines. Fig. 3A shows that the p50 subunit presented the highest activity in all the samples tested and correlated with both c-FLIP mRNA expression and sensitivity to TRAIL (see above). The p52 factor appeared to be activated only in the EBV-positive cell lines, JVM-2 and Granta-519 (15), and the other factors, p65, c-Rel, and RelB, remained poorly activated in all cell lines. Prompted by the potential impact of p50 transcriptional activity on the regulation of c-FLIP expression in MCL cells, and to increase MCL sensitivity to TRAIL, we further analyzed the effect of TRAIL, combining with inhibitors of the NF-{kappa}B canonical pathway that recruits p50. We compared the effect of IKK complex inhibitors, BMS-345541 and BAY-117082, and the proteasome inhibitor bortezomib, on the TRAIL-sensitive Jeko and TRAIL-resistant JVM-2 cell lines. As shown on Fig. 3B, low doses of BAY-117082 (0.5 and 1 µM), BMS-345541 (0.5–2 µM), and bortezomib (5 and 10 nM), though no cytotoxic in Jeko cells, synergistically increased TRAIL cytotoxicity, as determined by the Chou and Talalay’s method (combination index <0.7). Conversely, JVM-2 cells remained resistant to TRAIL even when cells were pretreated with increasing doses of bortezomib or BAY-117082. However, BMS-345541 at 2 µM was the only IKK inhibitor able to initiate TRAIL-induced cell death in this cell line (Fig. 3B, lower panel).


Figure 3
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  		FIGURE 3. Selective inhibition of NF-{kappa}B signaling increased the sensitivity of MCL cells to TRAIL. A, DNA-binding activity of the five NF-{kappa}B factors p50, p52, p65, c-Rel, and RelB was measured in nuclear extracts from MCL cell lines as described in Materials and Methods. Values are expressed in relative luminescent units (RLU) as the mean of duplicates. B, JVM-2 and Jeko cells were pretreated for 3 h with increasing doses of bortezomib or IKK inhibitors BAY-117082 or BMS-345541, followed by a 16 h treatment with 5 ng/ml (Jeko) or 100 ng/ml (JVM-2) TRAIL. Cell viability was evaluated by means of annexin V labeling. Arrows point out the most efficient synergistic combination of TRAIL with bortezomib (10 nM), BAY-117082 (1 µM), and BMS-345541 (2 µM). C, Western blot analysis of the expression of TRAIL-related and NF-{kappa}B-regulated proteins in Jeko and JVM-2 cells treated for 6 h with 10 nM bortezomib (bz), 1 µM BAY-117082 (BAY), or 2 µM BMS-345541 (BMS). D, Flow cytometry analysis of TRAIL receptors in Jeko and JVM-2 cells, either untreated (gray histograms) or treated as in C (dark histograms). Open areas correspond to unlabeled cells.

 
We next assessed the expression of several NF-{kappa}B target genes related to TRAIL signaling, after treatment of Jeko and JVM-2 cells with these three compounds. Fig. 3C shows that bortezomib treatment induced an increase in c-FLIPS expression, and that this effect was most pronounced in JVM-2 than in Jeko cells. In parallel, the two IKK inhibitors reduced c-FLIP levels, being this down-regulation more evident in JVM-2 cells treated with BMS-345541. In these same cells, we also observed a slight decrease in XIAP expression (Fig. 3C). BMS-345541 also reduced DR4 expression in both cell lines, while the other IKK inhibitor BAY-117082 induced DR4 up-regulation in Jeko cells (Fig. 3D). These results demonstrate that BMS-34541 presents a considerable advantage on the inhibition of NF-{kappa}B-regulated factors that negatively regulate TRAIL apoptotic signaling in JVM-2 cells.

IKK inhibition activates the TRAIL apoptotic pathway at the DISC level

To confirm that BMS-345541 was able to sensitize MCL cells to TRAIL via reducing c-FLIP expression, we first investigated the effects of this inhibitor on c-FLIP transcription. After 6 h treatment with 2 µM of BMS-345541, we observed a dramatic decrease of c-FLIP mRNA expression in the three representative cell lines Jeko, Granta-519, and JVM-2 (Fig. 4A). Interestingly, the repression of c-FLIP transcription tightly correlated with the inhibition of the basal I{kappa}B phosphorylation in each cell line (Fig. 4B). Precipitation analysis revealed that the TRAIL-dependent DISC harbored significant reduction in c-FLIPS and c-FLIPL amounts in JVM-2 cells cotreated with BMS-345541 and Bio-TRAIL, when compared with cells incubated with Bio-TRAIL alone (Fig. 4C). The decrease in c-FLIP levels enabled caspase-8, but not FADD, recruitment and cleavage inside the complex. Consequently, while 2 µM BMS-345541 alone did not induce caspases activation, it activated both extrinsic (as revealed by caspase-8- and Bid cleavage) and intrinsic (including caspase-9, -3, Mcl-1, and PARP cleavage) apoptotic pathways in combination with TRAIL, in both Jeko and JVM-2 cell lines (Fig. 4D). Therefore, this combination allowed the release of the apoptotic mitochondrial factors cytochrome c and Smac/DIABLO to the cytosol (data not shown). Bortezomib also facilitated TRAIL-induced caspase signaling in the TRAIL-sensitive cell line Jeko, but not in the TRAIL-resistant JVM-2 (data not shown).


Figure 4
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  		FIGURE 4. Effect of the BMS-345541 inhibitor on TRAIL apoptotic signaling. A, c-FLIP mRNA levels was evaluated by real-time RT-PCR, in control and BMS-345541-treated Jeko, Granta-519, and JVM-2 cells. Results are given in arbitrary units, using cDNA from untreated JVM-2 cells as calibrator. B, Western blot analysis of I{kappa}B phosphorylation status (p-IkB) after a 6 h treatment of Jeko, Granta-519, and JVM-2 cells with 2 µM BMS-345541. C, Western blot analysis of c-FLIP, caspase-8, and FADD on TRAIL-attached (bound) and TRAIL-free (unbound) fractions from JVM-2 cells treated for 1 h with 500 ng/ml biotinylated TRAIL (Bio-TRAIL), with or without pretreatment with 2 µM BMS-345541. D, Expression patterns of TRAIL-mediated apoptotic signaling proteins in Jeko and JVM-2 cells treated for 16 h with 5 or 100 ng/ml TRAIL, respectively, with or without a 3 h preincubation with 2 µM BMS-345541. Total cell extracts were analyzed by Western blot, using actin and tubulin as loading controls. E, Effect of caspase-8/10 inhibition on BMS-345541 and TRAIL synergistic apoptotic signaling. Jeko cells were treated for 16 h with 5 ng/ml TRAIL and 2 µM BMS-345541, in the presence or the absence of 50 µM of the caspase-8/10 inhibitor Z-Ile- Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone (z-IETD.fmk; Calbiochem). Then, cells were analyzed for caspase-3 activation (upper panel, shown is the percentage of cells with activated caspase-3 (black histogram)) and caspase signaling (lower panel).

 
The caspase-8/10 inhibitor z-IETD.fmk reversed the effects of BMS-345541 on TRAIL-induced apoptosis in Jeko cells, as demonstrated by flow cytometry quantification of caspase-3 activated form, and immunoblot detection of caspase-8/-9, Bid, and PARP cleavage (Fig. 4D).

These results confirmed that BMS-345541-mediated inhibition of NF-{kappa}B activation results mainly in c-FLIP repression, enabling the formation of the TRAIL-dependent DISC and caspase-8-dependent apoptotic signaling.

I{kappa}B inhibition increases TRAIL cytotoxicity in MCL primary cells

To validate the above results with MCL primary cells, we tested the sensitivity to TRAIL on primary cultures from 10 MCL patients (Table I). Incubation of MCL cells with 100 ng/ml TRAIL for 16 h induced a decrease in cell viability of >25% in 7 of the 10 (70%) MCL primary cultures. Cells from patients 5, 7, and 6 were barely sensitive to TRAIL-mediated cytotoxicity, as well as normal PBLs (Fig. 5A). As observed with MCL cell lines, there was no correlation between sensitivity of MCL cells to TRAIL and 8p genomic loss (Table I). In an attempt to increase the cytotoxic effect of TRAIL, cells from three TRAIL-sensitive patients (1, 3, and 8) and two TRAIL-resistant ones (6 and 5) were pretreated with BMS-345541 before TRAIL addition. BMS-345541 treatment allowed for an increase in TRAIL cytotoxicity in the five MCL samples tested (Fig. 5B). TRAIL-resistant cells (patient 5) expressed a higher basal c-FLIP level than TRAIL-sensitive cells (3), and BMS-345541 was able to reduce c-FLIP expression in both cases. This effect was accompanied by a reduced phosphorylation of I{kappa}B, confirming the inhibition of NF-{kappa}B pathway (Fig. 5C). Thus, MCL primary cells responded to TRAIL in the majority of cases and this response was significantly increased by BMS-345541 that modulated both I{kappa}B phosphorylation status and c-FLIP intracellular levels. Combining TRAIL and BMS-345541 was not found to be cytotoxic in normal PBLs, indicating that the combination of TRAIL with IKK inhibition may be a specific target for tumor cells (Fig. 5B).


Figure 5
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  		FIGURE 5. I{kappa}B inhibition sensitizes MCL primary cells to TRAIL. A, Cells from 10 MCL patients (Table I) and from three healthy donors (PBLs) were cultured for 16 h with 100 ng/ml TRAIL. Then cells were labeled with Annexin VFITC and propidium iodide and analyzed by flow cytometry. Viability is expressed referred to untreated cells (100%). B, Control PBLs and cells from three TRAIL-sensitive (nos. 1, 3, and 8) and two TRAIL-resistant (nos. 6 and 5) patients were preincubated or not for 3 h with 2 µM BMS-345541 and then treated as in A. Cell death is expressed referred to untreated cells. ***, p < 0.001 with Student’s t test. C, Whole protein extracts from a representative TRAIL-sensitive and TRAIL-resistant patient (nos. 3 and 5, respectively), were subjected to SDS-PAGE and analyzed for c-FLIP and phospho-I{kappa}B (p-IkB) expression. Actin levels were checked to confirm equal loadings.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
MCL is an aggressive B cell lymphoma with poor prognosis and resistance to current chemotherapy regimens (1). Novel, rationally based, and tumor-specific treatments are therefore needed to improve patient outcome. Consequently, agents such as TRAIL that can selectively target tumor cells without causing toxicity to surrounding tissue are crucial in clinical approaches to MCL treatment.

This study provides evidence that the TRAIL apoptotic pathway is functional in the majority of primary and established MCL cell lines. We also demonstrate that sensitivity to TRAIL in MCL cells is not linked to TRAIL receptor expression. TRAIL-induced apoptosis has recently been described as dependent on TRAIL-RI and/or TRAIL-R2 dosage in B cell lymphomas, and associated with the 8p21.3 deletion, frequently observed in the leukemic form of MCL (16). Our results show that 8p genomic loss is associated neither with response to TRAIL, with mRNA levels, nor with the membrane expression of the TRAIL receptors. Moreover, analysis of DR4 and DR5 expression reveals that cell lines with the lowest sensitivity to TRAIL (Rec-1, Granta-519, and JVM-2) express the highest amounts of receptors. A lack of correlation between sensitivity to TRAIL cytotoxic effect and membrane expression of its receptors has been described in other models (21, 22). Although a frequent polymorphism affects the TRAIL receptor locus in MCL cells, no mutations are observed in their death domain (23). Thus, in MCL cells resistant to TRAIL, defects in TRAIL-dependent apoptotic signaling might occur downstream of the ligand-receptor interaction.

Overexpression of some IAPs, such as XIAP, or enhanced Akt activity has been reported to confer resistance to TRAIL-induced apoptosis in cancer cells (24, 25, 26). TRAIL sensitivity can also be modulated by the expression levels of c-FLIP, Bcl-xL, p53, protein kinase C, MAPK, or c-myc (27, 28). Among these possible regulatory factors, our results identify c-FLIP as an important regulator of MCL cells sensitivity to TRAIL. c-FLIP expression correlates with TRAIL sensitivity in both MCL cell lines and primary tumor cells. Indeed, as observed in other models, in response to doxorubicin (29), during cell differentiation (30) or phosphorylation (31), down-regulation of functional c-FLIP appeared to be a prerequisite to increase the sensitivity of MCL cells to TRAIL, particularly in cases with high c-FLIP basal level, such as in JVM-2 cells. Because c-FLIP is known to be regulated by NF-{kappa}B, and considering that NF-{kappa}B is constitutively up-regulated in MCL cells (32), we addressed NF-{kappa}B activation status in MCL cell lines. Transcriptional activity of the NF-{kappa}B factor p50 is found to be closely related to c-FLIP expression, and that inhibition of the canonical NF-{kappa}B pathway allows all primary MCL cells and MCL cell lines to undergo TRAIL-induced apoptosis. Our study suggests that the use of either IKK inhibitors or bortezomib to sensitize cells to TRAIL depends on the basal c-FLIP levels. Furthermore, c-FLIP accumulates after bortezomib treatment and could interfere with TRAIL-mediated cell death, as observed in JVM-2 cells, because the protein is subject to ubiquitination and proteasomal degradation (33). Despite this, bortezomib-mediated proteasome inhibition remains an efficient way to sensitize MCL cells with low c-FLIP levels to TRAIL as observed in Jeko cells, as it has been previously reported (34, 35). The synergistic effect observed between the two compounds in Jeko cells was mainly based on caspase activation, because the pan caspase inhibitor z-VAD.fmk reversed virtually all the beneficial effects of bortezomib on TRAIL-mediated apoptotic signaling (data not shown).

The selective inhibitor of the catalytic subunit of IKK, BMS-345541 (36), in contrast to bortezomib, allows TRAIL to fully signal apoptosis in both TRAIL-resistant and TRAIL-sensitive MCL cells, by decreasing c-FLIP expression and consequently activating the upstream components of the TRAIL apoptotic pathway. However, in TRAIL-resistant cells (JVM-2, patient 5) that express high levels of c-FLIP, treatment with BMS-345541 consistently reduces c-FLIP expression, although protein levels do not reach those observed in BMS-345541-treated TRAIL-sensitive cells (Jeko and patient 3). This may explain why the IKK inhibitor preferentially shows a pronounced synergistic activity in cells with low levels of c-FLIP and sensitive to TRAIL. Furthermore, our results show that BMS-345541 reduces not only c-FLIP levels, but also DR5 expression. Indeed, the expression of DR5 has also been described as being regulated by NF-{kappa}B (37). The decrease in DR5 expression induced by BMS-345541 seems not to interfere with its sensitizing effect on TRAIL-induced cell death, suggesting that this receptor might not be required for TRAIL apoptotic signaling in MCL cells. These results agree with recent studies describing DR4 as the principal TRAIL receptor implicated in TRAIL-mediated apoptosis in lymphoid malignancies (38, 39). Accordingly, TRAIL-dependent apoptosis is not activated after incubation of MCL cells with a recombinant TRAIL which preferentially binds to the DR5 receptor (Apo2L; Genentech) (7), even in the presence of BMS-345541 (data not shown).

Considering that both TRAIL and BMS-345541 have already demonstrated selective cytotoxicity against malignant cells (5, 6, 40), combining TRAIL, or anti-DR4 Abs, with pharmacological inhibitors of IKK signaling may represent an attractive model for the design of a new and rational combination therapy for MCL.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was financially supported by Fondo de Investigaciones Sanitarias FIS03/0398 (to D.C.), the Lymphoma Research Foundation, European Commission Contracts SLMM-CT-2004-503351, Redes temáticas de Centros: Genómica del cancer (03/10) and Red Estudio de neoplasias linfoides (03/179), Instituto de Salud Carlos III. G.R. and P.P.-G. hold postdoctoral contracts from the c-RED program (Generalitat de Catalunya) and from the Juan de la Cierva program (Ministerio de Educación y Ciencia), respectively. M.L.-G. is the recipient of a predoctoral fellowship from Generalitat de Catalunya. Back

2 Address correspondence and reprint requests to Dr. Dolors Colomer, Hematopathology Unit, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. E-mail address: dcolomer{at}clinic.ub.es Back

3 Abbreviations used in this paper: MCL, mantle cell lymphoma; DR, death receptor; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; DcR, decoy inhibitory receptor; c-FLIP, cellular FLIP; c-FLIPS, c-FLIP short form; c-FLIPL, c-FLIP long form; IKK, I{kappa}B kinase; IAP, inhibitor of apoptosis protein; XIAP, X-chromosome linked IAP; siRNA, small-interfering RNA. Back

Received for publication June 1, 2006. Accepted for publication November 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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