Background
Neuroblastoma (NBL) is a solid tumor that arises from neuronal crest cells of the sympathetic nervous system. The most common form of cancer in infancy, NBL causes 15% of cancer-related deaths in children. The tumors have remarkable heterogeneity, which become evident in the clinic where patients can show spontaneous regression or rapid and fatal tumor progression. Over the years, significant advances have been made in the treatment of low- and intermediate-risk patients, thus allowing reaching high survival rates; however, the 5-year survival rate of patients in the high-risk group is still below 50% [
1-
3].
High-risk NBLs are treated with surgery, chemotherapy, radiotherapy, and/or the use of biological agents. Most of the therapeutic strategies used in NBL interfere with cell cycle progression and DNA synthesis or function, thereby causing DNA damage and the induction of apoptosis through the intrinsic and extrinsic apoptotic pathways [
4].
The extrinsic or Death receptor (DR) pathway is activated by cell surface receptors of the tumor necrosis factor receptor (TNFR) family, which includes receptors for TNFα, FasL, and TNF-related apoptosis-inducing ligand (TRAIL) [
5-
7]. These receptors contain a death domain in their cytosolic tail which upon receptor activation leads to context-dependent outcomes such as apoptosis, necroptosis, or pro-survival signaling. The targeting of DR signaling has been proposed and studied for the treatment of various types of cancers [
8-
10]. For NBL tumors, this strategy has been largely disregarded, possibly because caspase-8 silencing occurs in 50-70% of all human NBLs [
11-
14]. However, a significant group of NBL patients do express caspase-8 and could benefit from treatments that induce DR activation. Given that TNFα is able to upregulate Fas expression in human cancer cell lines and sensitize them to FasL-induced cell death [
15-
17], we sought to investigate whether TNFα and FasL combination could be therapeutically relevant in NBL.
We found that TNFα treatment primes a subset of NBLs for FasL-induced cell death by triggering the NF-κB-mediated upregulation of Fas. Moreover, TNFα pre-treatment increased cisplatin- and etoposide-induced caspase-8 cleavage and cell death in NBL cells that express both Fas and caspase-8. Our findings suggest that selected NBL patients could benefit from treatments that target TNFR1 and upregulate Fas expression.
Discussion
Many patients with high-risk NBL tumors continue to have a poor prognosis. Consequently, ongoing efforts are being channeled into the development of new treatments or the discovery of therapeutic agents that can increase the efficacy of current clinical regimes—cisplatin and etoposide being examples of such drugs [
2]. Here we describe that the activation of TNFR1 increases susceptibility to FasL-, cisplatin- and etoposide-induced cell death through the NF-κB -mediated upregulation of Fas, a target that has received little attention for NBL therapies. The newly synthesized Fas is exposed to the cell surface and incorporated into the DISC complex upon ligand binding, thereby triggering the activation of caspases and inducing apoptotic cell death.
Soluble TNFα exerts its effects through the binding and activation of the ubiquitously expressed TNFR1 receptor [
5-
7,
18,
19]. Depending on the cellular context, TNFα stimulation induces apoptosis, necroptosis, or pro-survival signaling through the activation of caspases, kinases, and transcription factors such as NF-κB [
5-
7,
21]. For NF-κB activation, TNFR1 binds the adaptor protein TRADD through interaction with its death domain. This interaction allows the recruitment of the adapter protein RIP1 and the E3 ligases TRAF2/5 and cIAP1/2, thereby inducing the ubiquitination of RIP1. This shapes the platform for recruitment and activation of the IKK complex that induces phosphorylation of the cytoplasmic NF-κB inhibitor IκBα, thereby targeting it for ubiquitination and subsequent proteasomal degradation. Degradation of IκBα mediates the release of NF-κB and allows its translocation to the nucleus where it can induce gene transcription. According to our data and data from others,
FAS is amongst the genes that can be induced by NF-κB. Chan
et al. and Liu
et al. have previously identified the p65/RelA binding site in the Fas promoter and confirmed TNFα-induced NF-κB -mediated upregulation of Fas [
32,
33]. Here, we were able to demonstrate the NF-κB -mediated regulation in NBLs and discarded regulation of Fas expression by other pathways known to be activated by TNFR1 (i.e. ERK1/2, PI3K, and JNK).
Given the participation of the Fas/FasL system in the mechanisms of cell death caused by DNA-damaging agents such as cisplatin and etoposide [
26,
27], we studied the possibility of improving the efficacy of these drugs by combined treatment with TNFα. Our results showed that TNFα pre-treatment increased cisplatin- and etoposide-induced cell death in two of the four NBL cell lines studied. Similarly, Benedetti
et al. reported that TNFα acts in synergy with cisplatin in renal proximal tubular cells, inducing an increase in cell death by prolonging JNK activation and inhibiting NF-κB translocation to the nucleus [
34,
35]. However, our data indicate that the TNFα-induced priming for cisplatin- and etoposide-induced cell death depends on NF-κB -mediated induction of Fas expression and caspase-8 cleavage.
Remarkably, not all the NBL cell lines studied were primed by TNFα for cisplatin- and etoposide-induced cell death. To predict the benefit of the TNFα combination therapy, we analyzed the expression of Fas and the modulation thereof by TNFα in a set of eight NBL cell lines. In four of the eight NBL cell lines, TNFα upregulated Fas expression. Furthermore, we observed that only the cell lines that showed TNFα-induced upregulation of Fas expression also displayed TNFα-induced priming to FasL-, cisplatin-, and etoposide-induced cell death. The cell lines that showed TNFα-induced priming also displayed Fas and caspase-8 expression, whereas cell lines that were not primed by TNFα showed the expression of only one of the two proteins. The response to TNFα treatment was not related to other frequent NBL alterations, such as MYCN amplification or p53 functional status (see Table
1).
Table 1
Neuroblastoma characteristics and their modulation by TNFα
Expression
|
Fas
| + | + | +/− | +/− | - | - | + | ++ |
Caspase-8
| ++ | ++ | +/− | - | +/− | +/− | - | ++ |
p53
| | | | | | | | |
TNFα-induced
|
Fas
| ++ | + | + | - | - | - | - | ++ |
Sensitization to etoposide/cisplatin
| ++ | + | NA | - | - | NA | NA | NA |
FasL-induced cell death
|
UT
| + | +/− | - | - | - | - | - | ++ |
TNFα
| +++ | +++ | + | - | - | - | - | +++ |
The mechanism by which Fas is silenced in NBL and why some cell lines do not respond to the TNFα-induced Fas regulation remains to be clarified. In the NBL cell lines addressed, we confirmed NF-κB activation after TNFα treatment and detected the induction of other known NF-κB target genes, such as cIAP2 and Bcl-2 [
24,
28]. One possible mechanism to explain this lack of Fas induction is that TNFα treatment stimulates the formation of different NF-κB heterodimers or NF-κB was post-transcriptionally modified, which may drive specific gene expression [
42]. An alternative mechanism to account for the incapacity of TNFα to induce Fas expression can be found at the level of epigenetic regulation of the Fas gene. Methylation of the Fas promoter has been reported in various types of tumors, including NBL [
43-
45]. IFNγ has been shown to restore caspase-8 and Fas expression in NBL cells [
29-
31,
46,
47] and to render them sensitive to FasL treatment. Consequently, IFNγ may also prime caspase-8- or Fas-deficient NBL cells for the TNFα combination therapy. Indeed, we confirmed that IFNγ primes these NBL cells for FasL-induced cell death. However, IFNγ treatment did not sensitize all the NBL cell lines to the TNFα-induced upregulation of Fas. These findings suggest that the expression of Fas in NBLs is regulated at various levels and that it differs between NBLs.
Recent studies have described the benefits of TNFα in combination with doxorubicin [
48] or melphalan [
49] for the treatment of solid tumors. Due to its low toleration in systemic treatment, various TNFα fusion proteins have been developed for localized treatment [
50], some of which show promise and have entered clinical trials [
49,
51,
52]. These findings break ground for the use of TNFα in the treatment of NBL in combination with cisplatin and etoposide.
Our results suggest that NF-κB -mediated upregulation of Fas by TNFα could be a new approach for the treatment of NBL patients. These findings are in contradiction to the current dogma in which NF-κB inhibition is seen as a strategy for cancer treatment, since NF-κB has been implicated in promoting cancer initiation, development, and metastasis [
53,
54]. NF-κB activation is known to promote cell survival by upregulating anti-apoptotic proteins, such as Bcl-2, c-FLIP, and cIAP2 thereby inhibiting DR-induced apoptosis [
24,
25,
28]. However, NF-κB is also able to promote apoptosis through the induction of pro-apoptotic proteins, such as Fas [
32,
33], Bax [
55], DR5 [
56], and DR6 [
57]. Our study supports the evidence that NF-κB triggers pro-apoptotic signaling in a subset of NBL cells through Fas upregulation, which tips the scale towards apoptotic cell death.
Methods
Reagents
Unless stated otherwise, all biochemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Fc:hFasL was a generous gift of Dr. Pascal Schneider (University of Lausanne, Epalinges, Switzerland). Recombinant human TNFα and IFNγ were supplied by Biotrend (Köln, Germany). PD98059, SP600125, BAY 11–7082, Z-IETD-FMK, and Q-VD-OPH were purchased from Merck Millipore (Billerica, MA, USA).
Cell culture
The human NBL cell lines SK-N-AS, LAI-5S, IMR32, SK-N-BE(2), and SH-SY5Y and the renal epithelial cell line HEK293T were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% or 15% (SH-SY5Y) heat-inactivated FBS (FBSi, Thermo Fisher Scientific). The NBL cell lines SK-N-SH and CHLA90 were cultured in IMDM (Thermo Fisher Scientific) supplemented with 20% FBSi. The NBL cell line Tet21N was maintained in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FBSi, 25 mM HEPES (Thermo Fisher Scientific), 200 μg/ml geneticin (G418), 0.5 μg/ml amphotericin B, and 10 μg/ml hygromycin B. Cell culture media was supplemented with 100U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific). Cultures were maintained at 37°C in a saturated atmosphere of 95% air and 5% CO2. CHLA90 cells were acquired from the Children’s Oncology Group Cell Line repository. SK-N-BE(2) and LAI-5S cells were from the Public Health England Culture Collections (Salisbury, UK). Tet21N cells were a kind gift from Dr. Manfred Schwab (DKFZ, Heidelberg, Germany). All other cell lines were acquired from the American Type Tissue Collection (ATCC, Manassas, VA, USA).
Hoechst staining
After the indicated treatments, cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton™ X-100, and stained with 0.05 μg/ml Hoechst 33342. Cell death was assessed by counting viable and dead cells, by discriminating condensed and fragmented nuclei (apoptotic nuclear morphology type II), as described by Yuste
et al. [
58]. Quantification was performed in blind testing, and at least 500 cells were counted per condition.
Caspase activity
After the indicated treatments, cells were harvested, washed with ice-cold PBS, lysed in caspase activity buffer (20 mM HEPES-NaOH, pH7.2, 10% sucrose, 150 mM NaCl, 5 mM EDTA, 1% Igepal CA-630, 0.1% CHAPS, and 1× EDTA-free Complete protease inhibitor mixture), and insoluble fractions were removed by centrifugation. The protein concentration of the lysate was quantified using the Lowry-based DC protein assay (Biorad, Hercules, CA, USA). Next, caspase activity was assessed by incubating 10 μg protein at 37°C in caspase activity buffer supplemented with 10 mM DTT and 50 μM of the fluorogenic substrate Z-IETD-Afc for caspase-8 activity or Ac-DEVD-Afc for caspase-3/7 activity (Merck Millipore). Caspase activity was assessed in a fluorometer using excitation and emission wavelengths of 405 nm and 535 nm, respectively.
Calcein AM
After the indicated treatments, cells were incubated for 1 h at 37°C with 1 μM Calcein AM (Merck Millipore) diluted in DPBS (Thermo Fisher Scientific). Fluorescence was then assessed in a fluorometer using excitation and emission wavelengths of 485 nm and 535 nm, respectively.
qRT-PCR
After treatment, cells were harvested, washed with ice-cold PBS, and RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Next, the RNA was retrotranscribed to cDNA using the High Capacity RNA-to-cDNA™ Kit (Thermo Fisher Scientific) and subjected to PCR analysis using Taqman® probes and Universal PCR Master Mix (Thermo Fisher Scientific). Taqman® probes: Fas (Hs00531110_m1), Caspase-8 (Hs01018151_m1), FADD (Hs00538709_m1), RIP1 (Hs00169407_m1), FasL (Hs00181225_m1), c-FLIP (Hs01116280_m1), Bcl-2 (Hs00608023_m1), and 18S (Hs03928990_g1).
Cell surface biotinylation
Cell surface proteins were biotinylated, isolated, and collected by using the Pierce® Cell Surface Protein Isolation Kit (Thermo Fisher Scientific), following the manufacturer’s instructions, with the only exception of equalizing protein quantity and concentration before immunoprecipitation. Protein levels were determined by Western blot.
DISC immunoprecipitation
For Fas DISC analysis, cells were treated with Fc:hFasL (2.5 μg/ml) for 30 min. The cells were then washed with ice-cold PBS, harvested, and lysed in ice-cold Triton lysis buffer (NaCl 150 mM, EDTA 10 mM, Tris–HCl pH7.4 10 mM, 1% Triton™ X-100, 1x EDTA-free complete protease inhibitor cocktail (Roche, Basel, Switzerland)). After lysate clearance by centrifugation, Fc:hFasL was immunoprecipitated from the supernatant by incubation with protein G-Sepharose beads for 1 h on an orbital shaker at 4°C. Next, the beads were washed 5x with ice-cold Triton lysis buffer, and the immunocomplexes were collected with elution buffer (Citrate 0.1 M, pH2.5). The pH was adjusted by adding 1/6 neutralizing buffer (Tris HCl 1 M, pH8.5). Protein levels were determined by Western blot.
Western blot
Cells were harvested, washed with ice-cold PBS, and lysed in ice-cold Triton lysis buffer or boiling SET buffer (Tris–HCl pH7.4 10 mM, EDTA 1 mM, NaCl 150 mM, 1% SDS). Insoluble fractions were removed by centrifugation, and protein concentration of the supernatant was quantified. The cell lysates obtained (25 μg of protein) were resolved in SDS-polyacrylamide gels. Next, proteins were transferred onto PVDF Immobilon-P membranes (Merck Millipore) by electrophoresis. Membranes were blocked with 5% non-fat dry milk in 1× TBS and 0.1% Tween-20 and probed with the appropriate primary antibodies [anti-Fas (C-20), anti-FADD (S-18), anti-c-FLIPS/L(H-202), anti-IκBα (C-21), anti-cIAP2 (H-85) (Santa Cruz, Biotechnology, Santa Cruz, CA, USA), anti-α-Tubulin (Sigma-Aldrich), anti-Bcl-2 (Dako, Agilent Technologies, Santa Clara, CA, USA), anti-Caspase-3 and anti-Caspase-8 (Cell Signaling Technologies, Beverly, MA, USA)] and the corresponding peroxidase-conjugated secondary antibodies (Sigma-Aldrich).
Plasmids
The Super-repressor IκBα (SR) cDNA was subcloned from the validated pcDNA3 expression vector [
22,
59] into the lentiviral pWPI expression vector. SR was expressed under the control of the constitutively active EF-1 Alpha promoter.
Lentiviral production and cell infection
Lentiviruses were produced in HEK293T cells by Lipofectamine 2000 (Thermo Fisher Scientific) co-transfection of pWPI-derived constructs, pSPAX2, and pM2G in a 3:2:1 ratio, respectively. Cells were allowed to generate lentiviruses for 48 h, after which the lentivirus-bearing medium was collected and passed through a Whatman® 45 μm filter (GE Healthcare, Little Chalfont, UK). For infection, the lentivirus-bearing medium was added to the host cells in combination with 8 μg/ml polybrene. Infection efficiency was assessed by direct counting of GFP-positive cells, and infection was repeated until an efficiency of ≥95% was reached.
Flow cytometry
After the indicated treatments, cells were detached with cell dissociation buffer (PBS, 5 mM EDTA), harvested, washed 2× with ice-cold PBS and 1× with ice-cold FACS buffer (PBS, 2% FBSi, 0.02% sodium azide), and then incubated for 30 min on ice with a PE-conjugated monoclonal antibody against Fas or its matched isotype (Becton Dickinson, Franklin Lakes, NJ, USA). Thereafter, cells were washed 2× and resuspended in ice-cold FACS buffer. Fas expression was assessed by a FACSCalibur™ flow cytometer (Becton Dickinson).
Statistical analysis
All the experiments were repeated at least three times. Values are expressed as mean ± SD. Statistical significance was determined by one-way or two-way ANOVA using GraphPad Prism v5 (GraphPad Software, La Jolla, CA, USA).
Acknowledgements
We thank Dr. Pascal Schneider for providing the recombinant Fc:hFasL and Dr. Manfred Schwab for supplying us with the Tet21N cell line. This work was funded by the Spanish Government’s “Ministerio de Economía y Competitividad” (SAF2010-19953, SAF2013-47989-R, CIBERNED CB06/05/1104 and PIE13/00027, to JXC), the “Generalitat de Catalunya” (SGR2009-346, 2014SGR1609, to JXC), and the “Instituto de Salud Carlos III” (CP11/00052, RD12/0036/0016, to MFS) co-financed by the European Regional Development Fund (ERDF). KMOG is supported by a postgraduate fellowship which is part of the SAF2010-19953.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JXC, MFS, and KMOG designed the experiments. KMOG, PC, LP-F, JU, EC, JL-S, and RSM performed the laboratory work and collected the data. KMOG, JXC, and MFS analyzed and interpreted the data. KMOG, MFS, JXC, and BB-Z wrote the manuscript. The final manuscript was read and approved by all signing authors.