Inflammatory Cell Death, PANoptosis, Mediated by Cytokines in Diverse Cancer Lineages Inhibits Tumor Growth - PubMed (original) (raw)

Inflammatory Cell Death, PANoptosis, Mediated by Cytokines in Diverse Cancer Lineages Inhibits Tumor Growth

R K Subbarao Malireddi et al. Immunohorizons. 2021.

Abstract

Resistance to cell death is a hallmark of cancer. Immunotherapy, particularly immune checkpoint blockade therapy, drives immune-mediated cell death and has greatly improved treatment outcomes for some patients with cancer, but it often fails clinically. Its success relies on the cytokines and cytotoxic functions of effector immune cells to bypass the resistance to cell death and eliminate cancer cells. However, the specific cytokines capable of inducing cell death in tumors and the mechanisms that connect cytokines to cell death across cancer cell types remain unknown. In this study, we analyzed expression of several cytokines that are modulated in tumors and found correlations between cytokine expression and mortality. Of several cytokines tested for their ability to kill cancer cells, only TNF-α and IFN-γ together were able to induce cell death in 13 distinct human cancer cell lines derived from colon and lung cancer, melanoma, and leukemia. Further evaluation of the specific programmed cell death pathways activated by TNF-α and IFN-γ in these cancer lines identified PANoptosis, a form of inflammatory cell death that was previously shown to be activated by contemporaneous engagement of components from pyroptosis, apoptosis, and/or necroptosis. Specifically, TNF-α and IFN-γ triggered activation of gasdermin D, gasdermin E, caspase-8, caspase-3, caspase-7, and MLKL. Furthermore, the intratumoral administration of TNF-α and IFN-γ suppressed the growth of transplanted xenograft tumors in an NSG mouse model. Overall, this study shows that PANoptosis, induced by synergism of TNF-α and IFN-γ, is an important mechanism to kill cancer cells and suppress tumor growth that could be therapeutically targeted.

Copyright © 2021 The Authors.

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Figures

Figure 1:

Figure 1:. Concomitant treatment of TNF-α and IFN-γ triggers robust cell death in NCI-60 colon cancer cells

(A) Heat map representing the levels of inflammatory cytokines in the tumor microenvironment relative to levels in healthy tissue. (B-C) Quantification of the cell death in HCT-116 colon cancer cells treated with media or the indicated cytokines and assessed in culture 48 h post-stimulation. “Cocktail-1” (Cockt-1) is a combination prepared by mixing all the individual cytokines used in this panel (TNF-α, IFN-γ, IL-1α, IL-1β, IL-18, IL-2, IL-6, IL-8 and IL-15); Cocktail-2 (Cockt-2) is same as Cockt-1, except that it lacks TNF-α and IFN-γ (contains L-1α, IL-1β, IL-18, IL-2, IL-6, IL-8 and IL-15). (D) Representative images of cell death detected by IncuCyte imaging analysis of cancer cells treated with TNF-α and IFN-γ at 48 h post-treatment. Scale bar, 50 μM. (E) Time-course analysis of cell death of cancer cells treated with TNF-α alone, IFN-γ alone, or TNF-α plus IFN-γ, assessed over the course of 48 h post-stimulation. Data are representative of three independent experiments (B-E). Data are presented as the mean ± SEM (B,C,E). ****P < 0.0001. Analyses were performed using the t test (B-C) or the two-way ANOVA (E).

Figure 2:

Figure 2:. TNF-α and IFN-γ treatment triggers PANoptotic cell death in human cancer cells

(A-C) Western blot analysis of PANoptosis components in HCT-116 colon cancer cells treated with cytokines as indicated and assessed in culture at 48 h post-stimulation. (A) Western blot analysis of the pyroptosis markers: pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) gasdermin D (GSDMD), and pro- (P53) and activated (P34) gasdermin E (GSDME). (B) Western blot analysis of the apoptosis markers: pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17), and pro- (P35) and cleaved caspase-7 (CASP7; P20). (C) Western blot analysis of necroptosis components: total MLKL (T-MLKL), MLKL oligomers, and total RIPK3 (T-RIPK3). (D-F) Western blot analysis of PANoptosis components in NCI-60 colon cancer cells treated with cytokines as indicated and assessed in culture at 48 h post-stimulation. (D) Western blot analysis of the pyroptosis markers: pro- (P45) and activated (P20) CASP1, pro- (P53) and activated (P30) GSDMD, and pro- (P53) and activated (P34) GSDME. (E) Western blot analysis of the apoptosis markers: pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17), and pro- (P35) and cleaved CASP7 (P20). (F) Western blot analysis of necroptosis components: T-MLKL, MLKL oligomers, and T-RIPK3. Western blot of β-Actin was used as loading control. Asterisks indicate non-specific bands. Data are representative of at least three independent experiments (A-F).

Figure 3:

Figure 3:. TNF-α and IFN-γ treatment triggers IRF1-dependent PANoptotic cell death

(A) Western blot analysis of IRF1 and STAT1 proteins in _IRF1_−/− and the corresponding control IRF1+/+ HCT-116 colon cancer cells, after treatment with indicated cytokines for 48 h. (B) Representative images of cell death detected by IncuCyte image analysis of indicated HCT-116 cells, treated with TNF-α and IFN-γ at 48 h post-treatment. Scale bar, 50 μM. (C) Quantitative real-time analysis of cell death in wild-type and _IRF1_-deficient HCT-116 colon cancer cells co-treated with TNF-α and IFN-γ. (D-F) Western blot analysis of PANoptosis components in wild-type and _IRF1_-deficient HCT-116 cells treated with TNF-α and IFN-γ for 48 h. (D) Western blot analysis of the pyroptosis markers: pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) gasdermin D (GSDMD), and pro- (P53) and activated (P34) gasdermin E (GSDME). (E) Western blot analysis of the apoptosis markers: pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17), and pro- (P35) and cleaved caspase-7 (CASP7; P20). (F) Western blot analysis of necroptosis components: total MLKL (T-MLKL), total RIPK3 (T-RIPK3), and MLKL oligomers. Western blot of β-Actin was used as loading control. Asterisks indicate non-specific bands. Data are representative of at least 3 independent experiments (A-F). Data are presented as the mean ± SEM (C). ****P < 0.0001. Analysis was performed using the two-way ANOVA (C).

Figure 4:

Figure 4:. JAK inhibition, but not nitric oxide inhibition, prevents the TNF-α and IFN-γ-dependent cancer cell death

Representative images (A) or time course analyses (B) of cell death detected by IncuCyte imaging of human HCT-116 colon cancer cells treated for 48 h with TNF-α and IFN-γ, in the presence or absence of the JAK1 inhibitor, baricitinib, or inducible nitric oxide inhibitor, 1400W. Scale bar, 50 μM. Data are representative of two independent experiments (A-B). Data are presented as the mean ± SEM (B). ****P < 0.0001. Analyses were performed using two-way ANOVA (B).

Figure 5:

Figure 5:. TNF-α and IFN-γ treatment suppresses transplanted tumor growth in vivo

(A) Diagram representing the NSG mouse model of tumorigenesis. (B) Time course analysis of tumor volume of transplanted COLO-205 cell tumors in NSG mice. (C) Weight of the tumors measured at the endpoint of the experiment, 28 post-inoculation and 16 days post-treatment with TNF-α and IFN-γ. (D) Representative images of the tumors that were collected at day 28 post-inoculation. (E) Depiction of the TNF-α plus IFN-γ–induced PANoptosis pathway based on the experimental findings from the current study. Data are presented as the mean ± SEM (B,C). *P < 0.05; ****P < 0.0001. Analyses were performed using the one-way ANOVA (B) or the t test (C).

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