Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency

Abstract

Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies.

Figure 1. Mitochondrial permeabilisation can engage necroptosis as a form of CICD

(A) SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-OPh (10 μM) and immunoblotted for PARP and β-Actin. Representative images from three independent experiments. (B) SVEC cells were treated for 72 h with ABT-737 (10 μM) +/- caspase inhibitor Q-VD-OPh (10 μM). For (B)(C)(D) and (E) cell viability was measured by flow-cytometry and PI exclusion. n=5 independent experiments. (C) SVEC cells stably expressing LZRS empty vector (vector) or LZRS-MCL-1 were treated for 72 h with ABT-737 (10 μM) +/- Q-VD-OPh (10 μM). n=3 independent experiments; mean values ± S.E.M (D) Control or RIPK3^SH SVEC cells were treated for 72 h with TNF (20 ng/ml) +/- caspase inhibitor zVAD-FMK (50 μM) and/or RIPK1 inhibitor necrostatin-1 (30 μM). n=3 independent experiments; mean values ± S.E.M (E) SVEC cells stably expressing pLKO empty vector (vector) or pLKO-shRIPK3 (RIPK3^SH) were treated for 72 h with ABT-737 (10 μM) +/- caspase inhibitor Q-VD-OPh (10 μM) and/or RIPK1 inhibitor necrostatin-1 (30 μM). n=3 independent experiments; mean values ± S.E.M. (F) BCL-xL dependent SVEC cells expressing empty vector (pLKO1) or pLKO-shAPAF-1 (APAF-1^SH) were treated for 24 h with ABT-737 (10 μM) +/- caspase inhibitor Q-VD-OPh (30 μM). For (F)(H)(I)(J) cell death was measured using an IncuCyte imager, measuring SYTOX Green uptake. n=3 independent experiments; mean values ± S.E.M. (G) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-OPh (30 μM) then immunoblotted for indicated proteins. Representative images from three independent experiments. (H) BCL-xL dependent SVEC cells stably expressing an empty vector (pLKO1) or pLKO1-shRIPK3 (RIPK3^SH) were treated with ABT-737 (10 μM) in the presence of caspase inhibitor Q-VD-OPh (30 μM) and/or necrostatin-1 (30 μM). A representative time-point shown (16 h). n=5 independent experiments; mean values ± S.E.M. (I) Control or MLKL-deleted BCL-xL dependent SVEC cells were treated with TNF (20 ng/ml) and zVAD-FMK (50 μM) +/- necrostatin-1 (30 μM). A representative time-point is shown (21 h). n=3 independent experiments; mean values ± S.E.M. (J) Control (vector^CRISPR) or MLKL-deleted (MLKL^CRISPR) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) together with Q-VD-OPh (30 μM) and/or necrostatin-1 (30 μM). A representative time-point is shown (21 h). n=3 independent experiments; mean values ± S.E.M.*p<0.05, **p<0.01, ***P<0.001; Holm-Sidak-corrected one way ANOVA (B), two-tailed unpaired t-test (C, D, E, H, I, J).

Figure 2. MOMP induces TNF-synthesis under caspase-deficient conditions

(A) SVEC cells were treated (72 h) with ABT-737 (10 μM) +/- Q-VD-OPh (10 μM) necrostatin-1 (30 μM) or Enbrel (50 μg/ml). Cell viability was measured by flow-cytometry (%PI⁺ cells). n=3 independent experiments; mean values ± S.E.M. (B) BCL-xL dependent SVEC cells were treated for 22 h with ABT-737 (10 μM) together with Q-VD-OPh (10 μM) or necrostatin-1 (30 μM) +/- Enbrel (50 μg/ml). cell death was measured using an IncuCyte imager, measuring SYTOX Green uptake. n=3 independent experiments; mean values ± S.E.M (C) BCL-xL dependent control or RIPK3 shRNA SVEC cells were treated as indicated with ABT-737 (10 μM), Q-VD-OPh (30 μM) and/or necrostatin-1 (30 μM), in the presence of TNF (20 ng/ml). Cell death was measured by IncuCyte imager, using SYTOX Green uptake, a representative time-point is shown (16 h). n=3 independent experiments; mean values ± S.E.M. (D) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-OPh (30 μM) and Tnf expression was measured by qRT-PCR. Data represent mean of triplicate samples and is representative of three independent experiments. (E) Control (vector^CRISPR) or BAX/BAK deleted BCL-xL dependent SVEC cells (BAX/BAK^CRISPR) were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) then Tnf expression was measured by qRT-PCR. Data represent the mean of triplicate samples and are representative of three independent experiments. (F) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) together with Q-VD-OPh (30 μM). Media TNF levels were measured by ELISA. n=3 independent experiments; mean values ± S.E.M. (G) BCL-xL dependent SVEC cells expressing APAF-1 shRNA (APAF-1^SH) were treated with ABT-737 (10 μM) and Tnf expression was measured by qRT-PCR. (H) Control or Caspase-9 deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Tnf expression was measured by qRT-PCR. (I) BCL-xL dependent E1A/Ras transformed MEFs were treated as in (D) and Tnf expression was measured by qRT-PCR. For (G)(H)(I) data represent the mean of triplicate samples and are representative of three independent experiments. *p<0.05, **p<0.01, ***P<0.001; two-tailed unpaired t-test (A, B) Holm-Sidak-corrected one way ANOVA (F). Statistical source data can be found in Supplementary Table 5.

Figure 3. Mitochondrial permeabilisation activates NF-κB

(A) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-Oph (30 μM) for 1 h, immunostained for p65 and analysed by confocal microscopy. TNF (20 ng/ml) was used as positive control for p65 nuclear translocation. Scale bar represents 30μM. Representative images from three independent experiments. (B) Quantification of cells (from A) displaying nuclear p65, minimum 300 cells were counted per condition. Ctrl: n=15 individual fields, ABT-737: n=15 individual fields, ABT-737/QVD: n=18, individual fields TNF: n=15 individual fields ± SEM. (C) BCL-xL dependent SVEC cells deleted for BAX and BAK (BAX/BAK^CRISPR) or cells expressing empty vector (vector^CRISPR) were treated with ABT-737 (10 μM) +/- Q-VD-OPh (30 μM) for 6 h then immunostained for p65 and cytochrome c. Scale bar represents 30μM. Representative images from three independent experiments. (D) Quantification of cells in (C) displaying nuclear p65, minimum 300 cells were counted per condition. Vector^CRISPR-ctrl: n=15 individual fields, vector^CRISPR-ABT-737/QVD: n=16 individual fields, BAX/BAK^CRISPR-ctrl: n=15 individual fields, BAX/BAK^CRISPR -ABT-737/QVD: n=16 individual fields ± SEM. (E) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-OPh (30 μM) and blotted for p-IκBα, total IκBα and β-Actin. .Representative image of three independent experiments. (F) BCL-xL dependent SVEC cells transiently expressing two different NFκB reporters (PGL3-TNF reporter 1 or PGL3-NP3 reporter 2 were treated with ABT-737 (10 μM) +/- Q-VD-OPh (30 μM) for 3 h and luciferase reporter assay was performed. Data represent the mean of duplicate samples and are representative of three independent experiments. (G) BCL-xL dependent SVEC cells stably expressing IκB^SR or empty vector (PMX) were treated with ABT-737 (10 μM) together with Q-VD-OPh (30 μM) or TNF (20 ng/ml) for 1 h and then immunostained for p65, minimum 300 cells were counted per condition. Quantification of cells displaying nuclear p65 was performed. Vector-ctrl: n=15 individual fields, vector-ABT-737/QVD: n=18 individual fields, vector-TNF/QVD: n=16 individual fields, IκB^SR-ctrl: n=15 individual fields, IκB^SR-ABT-737/QVD: n=15 individual fields, IκB^SR-TNF/QVD: n=15 individual fields ± SEM. (H) BCL-xL dependent SVEC cells stably expressing IκB^SR or empty vector (PMX) were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) and Tnf expression was measured by qRT-PCR. Data represent the mean of triplicate samples and are representative of three independent experiments. *p<0.05, **p<0.01, ***P<0.001; Tukey-corrected one way ANOVA (B), Tukey-corrected two way ANOVA (D, G). Statistical source data can be found in Supplementary Table 5. Unprocessed original scans of blots are shown in Supplementary Figure 9.

Figure 4. MOMP activates NF-κB through IAP down-regulation and NIK activation

(A) Cell lysates from BCL-xL dependent SVEC cells treated with SMAC-mimetic (SM-83, 50 nM) or ABT-737 (10 μM) and Q-VD-OPh (30 μM, 8h) were immunoblotted for indicated proteins. Representative image of three independent experiments. (B) Control or BAX/BAK deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM). Cell lysates were immunoblotted for indicated proteins. Representative images of three independent experiments. (C) Control, NIK-deleted or cIAP2 overexpressing BCL-xL dependent SVEC cells were treated with ABT-737 (10μM) and Q-VD-OPh (30 μM, 6h) and immunostained for p65, minimum 300 cells were counted per condition. Quantification depicts the percentage nuclear p65 positive cells. Vector^CRISPR-ctrl: n=15 individual fields, vector^CRISPR-ABT-737/QVD: n=18 individual fields, NIK^CRISPR-ctrl: n=16 individual fields, NIK^CRISPR-ABT-737/QVD: n=18 individual fields, vector-ctrl: n=15 individual fields, vector-ABT-737/QVD: n=18 individual fields, cIAP2-ctrl: n=15 individual fields, cIAP2-ABT-737/QVD: n=20 individual fields ± SEM. (D) Control, NIK-deleted or cIAP2 overexpressing BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 6h. Tnf expression was measured by qRT-PCR. Data represent the mean of triplicate samples and are representative of three independent experiments. (E) Control or cIAP2 overexpressing BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) as indicated and immunoblotted for NIK and β-Actin. Representative image of three independent experiments. (F) Wild-type or SMAC/OMI^-/- E1A/ras transformed MEFs were treated with ABT-737 (10 μM) and UMI-77 (10 μM) and Q-VD-OPh (30 μM) for 4 h. Cell lysates were immunoblotted for indicated proteins. Representative image of three independent experiments. (G) Wild-type or SMAC/OMI^-/- E1A/ras transformed MEFs were treated with ABT-737 (10 μM), UMI-77 (10 μM) and Q-VD-OPh (30 μM)(8h) then immunostained for cytochrome c and p65. Cells that had undergone MOMP were scored for nuclear p65 translocation, minimum 300 cells counted per condition. WT and SMAC/OMI^-/--ctrl: n=15 individual fields, wt and SMAC/OMI^-/--ABT-737/UMI-77/QVD: n=18 individual fields ± SEM. (H) BCL-2 dependent HeLa cells or cells transiently expressing TRAF2 together with either wild-type XIAP or XIAP BIR3 mutant (D214S E314S) were treated with ABT-263 (10 μM) and Q-VD-OPh (30 μM) for 4 h. Cell lysates were blotted for XIAP and β-Actin. Representative image of three independent experiments. Densitometric quantification of Western blots was performed. n = 3 independent experiments; mean values ± S.E.M.. *p<0.05, **p<0.01, ***P<0.001; Tukey-corrected two way ANOVA (C, G, H). Statistical source data can be found in Supplementary Table 5. Unprocessed original scans of blots are shown in Supplementary Figure 9.

Figure 5. Mitochondrial permeabilisation initiates an NF-κB dependent pro-inflammatory response

(A) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8h. Conditioned media was analysed for cytokine detection using Luminex assay. Data represents the mean of duplicate samples and is representative of two independent experiments. (B) BCL-xL dependent SVEC cells stably expressing IκB^SR or empty vector (vector) were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8h. Conditioned media was then analysed for cytokine levels using Luminex assay. Data is the mean of duplicate samples and is representative of two independent experiments. (C) BCL-xL dependent SVEC cells stably expressing IκB^SR or empty vector (PMX) were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 6 h and mRNA levels of Mcp-1, Kc, Gm-csf and Tnf were measured by qRT-PCR. (D) BCL-xL dependent SVEC cells stably expressing IκB^SR or PMX empty vector (vector) with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8 h and Ifnβ, Irf7 and Oasl1 were measured by qRT-PCR. (E) BCL-xL dependent SVEC cells deleted for STING (STING^CRISPR) or cells expressing empty vector (vector^CRISPR), as well as ρ⁰ cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 6 h, immunostained for p65 and analysed by confocal microscopy. Quantification includes the percentage of cells with nuclear p65, minimum 300 cells (left graph) and 240 cells (right graph) counted per condition. Vector^CRISPR, STING^CRISPR-ctrl and ABT-737/QVD: n=15 individual fields, vector^CRISPR and STING^CRISPR-TNF: n=11 individual fields, untreated and EtBr-ctrl and ABT-737/QVD: n=22 individual fields, untreated and EtBr-TNF: n=9 individual fields ± SEM. (F) BCL-xL dependent SVEC cells deleted for STING (STING^CRISPR) or expressing empty vector (vector^CRISPR) and ρ⁰ SVEC were treated as indicated and cell lysates were immunoblotted for cIAP1, XIAP and β-Actin. Representative image from three independent experiments. (G) BCL-xL dependent SVEC cells deleted for STING (STING^CRISPR) or expressing empty vector (vector^CRISPR) were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 6 h and mRNA levels of Mcp-1, Kc, Gm-csf and Tnf were measured by qRT-PCR. For (C), (D)and (G) data represent the mean of triplicate samples and are representative of three independent experiments. *p<0.05, **p<0.01, ***P<0.001; Tukey-corrected two way ANOVA (E). Statistical source data can be found in Supplementary Table 5. Unprocessed original scans of blots are shown in Supplementary Figure 9.

Figure 6. MOMP dependent NF-κB activity promotes macrophage activation

(A) Bone marrow-derived macrophages (BMDMs) were incubated overnight with conditioned media (CM) from BCL-xL dependent SVEC cells that had been treated with ABT-737 (10 μM) – CM (APO)- or ABT-737 (10 μM) and Q-VD-OPh (30 μM) – CM (CICD)- for 8 h. Expression of indicated M1 and M2 markers was determined by qRT-PCR. (B) BMDMs were treated as in (A) and stained for either M1 (CD86⁺) or M2 (CD206⁺) surface markers. n=3 independent experiments; mean values ± S.E.M. (C) BMDMs were incubated overnight with conditioned media (CM) from vector or APAF-1 knockdown BCL-xL dependent SVEC cells that had been treated with ABT-737 (10 μM) for 8 h. qRT-PCR was performed on BMDMs to assess M1 marker expression. (D) BMDMs were incubated overnight with conditioned media (CM) from control or BAX/BAK deleted BCL-xL dependent SVEC cells that had been treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8 h. qRT-PCR was performed on BMDMs to assess M1 marker expression. (E) BMDMs were incubated overnight with conditioned media (CM) from vector or IκB^SR overexpressing BCL-xL dependent SVEC cells that had been treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8 h. qRT-PCR was performed on BMDMs to assess M1 marker expression. (F) BMDMs were incubated overnight with conditioned media (CM) from murine primary pancreatic tumour cells (Myc-PDAC) that had been treated for 24 h with ABT-737 (10 μM) +/- Q-VD-OPh (10 μM). M1/M2 status was assessed with qRT-PCR. (G) BMDMs were incubated overnight with conditioned media (CM) transferred from murine primary pancreatic tumour cells (Myc-PDAC) that have been treated for 24 h with ABT-737 (10 μM) +/- Q-VD-OPh (10 μM). The activation profile was measured by FACS, staining for either M1 surface marker (CD86⁺) or M2 surface marker (CD206⁺). n=3 independent experiments; mean values ± S.E.M. For (A)(C)(D)(E)(F) data represent the mean of triplicate samples and are representative of three independent experiments. *p<0.05, **p<0.01, ***P<0.001; Tukey-corrected one way ANOVA (B, G). Statistical source data can be found in Supplementary Table 5.

Figure 7. CICD displays enhanced anti-tumorigenic effects versus apoptosis

(A) Control (pLKO1) or APAF-1 knockdown (APAF-1^SH) BCL-2 dependent CT26 cells were injected subcutaneously into Balb/C mice (5x10⁵ cells/mouse). Following tumour formation, mice were treated with either vehicle or ABT-263 (100mg/kg) for 2 times over a 7-day period then sacrificed one-day post-final treatment. Serum levels of the indicated cytokines were measured by Luminex assay. n=3 independent experiments; mean values ± S.E.M. (B) Representative immunohistochemistry images (n=3 mice) of CD3 staining (T cells), taken from control (pLKO1) or pLKO1-shAPAF-1 (APAF-1^SH) BCL-2 dependent CT26 cell tumour sections, following vehicle or ABT-263 (100mg/kg) treatment, 2 times in a week. Scale bar represents 100μM. (C) Control (pLKO1) or pLKO1-shAPAF-1 (APAF-1^SH) BCL-2 dependent CT26 cells were injected subcutaneously into Balb/C mice. Following tumour formation, mice were treated with vehicle or ABT-263 for 2 times in a 7-day period and sacrificed one day post-final treatment. T cell tumour infiltration was measured by flow-cytometry (CD3⁺DAPI^- cells). Vector-vehicle and ABT-263, APAF-1^SH-vehicle: n=9 mice, APAF-1^SH-ABT-263: n=8 mice ± SD. (D) BCL-2 dependent CT26 cells (wt vs APAF-1^SH) were injected into mice and treated with either vehicle or ABT-263 (100mg/kg) 2 times/week for a total of two weeks. Tumour growth was determined every 2 days by measuring the tumour volume (mm³). WT-vehicle, APAF-1^SH-vehicle and ABT-263: n= 10 mice, wt-ABT-263: n=9 mice± SEM from one experiment, repeated independently. Arrows indicate time of treatment. (E) Individual mouse tumour growth from (D) is shown. Post-treatment, each individual tumour was normalized (%) to the initial volume (mm³) obtained the first day of treatment (100% normalized tumour volume). Arrows indicate time of treatment. *p<0.05, **p<0.01, ***P<0.001; Tukey-corrected two way ANOVA (C, D).

Figure 8. CICD anti-tumourigenic effects requires NF-κB and intact immunity

(A) Vector or APAF-1^SH BCL-2 dependent CT26 cells were injected into Balb/C mice. Tumour bearing mice were treated with vehicle or ABT-263 (4 times over 2 weeks). Mice were sacrificed at day 13 post-treatment and split into responder (R) and non-responder (NR) groups. Total macrophage tumour infiltration (F4/80⁺) and M1-like macrophage activation status (NOS2⁺, CD86⁺ and MHC-II⁺) was measured by flow cytometry. (B) Samples generated in 8A were analysed for tumour-infiltrating activated CD4⁺ T cells (IFNγ⁺). (C) Samples generated in 8A were analysed for tumour-infiltrating activated CD8⁺ T cells (IFNγ⁺). For A-C The number of live infiltrated immune cells were measured by FACS and subsequently normalized to total live tumour cells and tumour weight. Data represents the mean ± SD of tumours; n=6-9 mice per group (individual n number per sub-group is provided in the figure). (D) BCL-2 dependent APAF-1^SH CT26 cells were injected into NSG mice. Tumour bearing mice were treated with vehicle or ABT-263. Data represents the mean ± SD of tumour volume (mm³). Arrows indicate treatment points; n=10 mice per group. (E) BCL-2 dependent APAF-1^SH CT26 cells were injected into Balb/C mice. Tumour bearing mice were pre-treated with isotype control or anti-Thy.1. One day later mice were treated with vehicle or ABT-263 (black arrows indicate treatment points) together with isotype control or anti-Thy.1 (red arrows indicate treatment points). Tumour growth was monitored every other day. APAF-1^SH + Isotype ctrl + ABT-263: n=10 mice, APAF-1^SH + anti-Thy.1 + ABT-263: n=9 mice ± SEM. (F) BCL-2 dependent APAF-1^SH CT26 cells and expressing vector or NEMO shRNA were injected into mice. Tumour bearing mice were treated with vehicle or ABT-263. Individual mouse tumour growth is shown. Post-treatment, each tumour was normalized (%) to the initial tumour volume (each set at 100%). Arrows indicate time of treatment and asterisks tumours that grew over 500% of the initial volume. APAF-1^SH-vector + vehicle: n=9 mice, APAF-1^SH-vector + ABT-263: n=10 mice, APAF-1^SH-NEMO^SH + vehicle: n=8 mice, APAF-1^SH-NEMO^SH + ABT-263: n=8 mice. (G) In silico analysis of clinical data from renal cell carcinoma patients using the TCPA database. *p<0.05, **p<0.01, ***P<0.001; Dunn’s-corrected Kruskal-Wallis (A, B, C), Tukey-corrected two way ANOVA (E), Spearman’s rank test (correlation), Cox and Log-Rank test (Kaplan Meier curves) (G).