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Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin

Abstract

Pyroptosis is a form of cell death that is critical for immunity. It can be induced by the canonical caspase-1 inflammasomes or by activation of caspase-4, -5 and -11 by cytosolic lipopolysaccharide1,2,3. The caspases cleave gasdermin D (GSDMD) in its middle linker to release autoinhibition on its gasdermin-N domain, which executes pyroptosis via its pore-forming activity4,5,6,7,8,9. GSDMD belongs to a gasdermin family that shares the pore-forming domain4,6,10. The functions and mechanisms of activation of other gasdermins are unknown. Here we show that GSDME, which was originally identified as DFNA5 (deafness, autosomal dominant 5)11, can switch caspase-3-mediated apoptosis induced by TNF or chemotherapy drugs to pyroptosis. GSDME was specifically cleaved by caspase-3 in its linker, generating a GSDME-N fragment that perforates membranes and thereby induces pyroptosis. After chemotherapy, cleavage of GSDME by caspase-3 induced pyroptosis in certain GSDME-expressing cancer cells. GSDME was silenced in most cancer cells but expressed in many normal tissues. Human primary cells exhibited GSDME-dependent pyroptosis upon activation of caspase-3 by chemotherapy drugs. Gsdme−/− (also known as Dfna5−/−) mice were protected from chemotherapy-induced tissue damage and weight loss. These findings suggest that caspase-3 activation can trigger necrosis by cleaving GSDME and offer new insights into cancer chemotherapy.

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Figure 1: GSDME expression switches TNF-induced apoptosis to pyroptosis owing to cleavage by caspase-3.
Figure 2: Caspase-3 cleaves GSDME in vitro to release the pore-forming activity.
Figure 3: Chemotherapy drugs induce pyroptosis in GSDME-positive cancer cells.
Figure 4: GSDME determines pyroptosis in primary human cells in response to chemotherapy drugs.
Figure 5: GSDME mediates chemotherapy drug-induced toxicity in mice.

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Acknowledgements

We thank F. Wang and the NIBS transgenic facility for generating Gsdme−/− mice, E. Zhang and J. Sui for reagents, and K. Jiang, Z. Liu and L. Sun for technical assistance. This work was supported by the National Key Research and Development Project on Protein Machinery and its Control and Regulation of Biological Processes (2016YFA0501500) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020202).

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Authors

Contributions

Y.W., W.G. and F.S. conceived the study; Y.W. and W.G. performed cellular studies; W.G. and X.S. performed mouse experiments; X.S. and Y.W. performed in vitro experiments; W.L., J.D., H.H. and K.W. provided technical assistance; Y.W., W.G. and F.S. analysed the data; F.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Feng Shao.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Albert, V. Hornung and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Ectopic expression of GSDME in HeLa cells switches TNF-induced apoptosis to pyroptosis, and dosage effect of GSDME expression.

a, b, Morphological and flow-cytometry assays of GSDME-induced apoptosis-to-pyroptosis switch. GSDMD−/− HeLa cells stably expressing the caspase-3-sensitive GSDMDDEVD or GSDMA3 or GSDMD−/−CASP3−/− HeLa cells stably expressing GSDME were assayed and phase-contrast cell images are shown in a. GSDMD−/− HeLa cells expressing an empty vector or GSDME were stained with propidium iodide and annexin V-Alexa 647 for flow cytometry (b). c, Effects of the caspase-3-specific inhibitor zDEVD on GSDME-mediated pyroptosis in GSDMD−/− HeLa cells. ATP cell viability and LDH-release cell death are expressed as mean ± s.d. from three technical replicates. d, e, Immunoblotting assays of caspase-3 cleavage of GSDME in GSDMD−/− or GSDMD−/−CASP3−/− HeLa cells. Flag–GSDME or Flag–GSDME1 (wild-type or D256A mutant) were stably expressed in cells. f, g, Two pools of HeLa cells expressing low (GSDMELow) or high (GSDMEHigh) levels of GSDME were obtained by flow-cytometry sorting; the immunoblots in f show the expression of GSDME. Cells were labelled by ectopically expressed enhanced green fluorescent protein (eGFP). g, Time-lapse phase-contrast and fluorescent images of cells taken at the indicated time points after stimulation (scale bar, 20 μm). Real-time videos are included in Supplementary Videos 6, 7, 8. All cells were stimulated with TNF + CHX. Data shown are representative of at least three independent experiments.

Extended Data Figure 2 Sequence alignment of GSDME from human, mouse, zebrafish and lancelet.

The alignment was generated using the ClustalW2 algorithm and presented using ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Identical residues are indicated by the dark red background and conserved residues are in red text. All GSDMEs share a gasdermin-N domain at the N terminus and a gasdermin-C domain at the C terminus, linked by a hinge loop. Zebrafish has two GSDMEs, GSDME1 and GSDME2 (previously known as DFNA5a and DFNA5b). The black box marks the caspase-3 cleavage motif in human and mouse GSDME and zebrafish GSDME1. No evident caspase-3-recognition motif could be found in the inter-domain linker regions of zebrafish GSDME2 or lancelet GSDME.

Extended Data Figure 3 Caspase-3 cleaves GSDME in vitro to release the pore-forming GSDME-N domain, which can trigger pyroptosis.

ac, In vitro cleavage of recombinant human GSDME or other caspase substrates by the p20/10 active form of various caspases (rCASPs). RhoGDI, GSDMD and BID are known substrates of caspase-3, caspase-1 and caspase-8, respectively. d, e, Lipid-binding and liposome-leakage activities of caspase-3-cleaved GSDME. GSDME-(N+C) is the noncovalent complex of GSDME-N and GSDME-C following in vitro caspase-3 cleavage of full-length GSDME (GSDME-FL). Mouse GSDME and zebrafish GSDME1 were assayed and GSDMD was included as a positive control. The indicated gasdermin proteins were incubated with liposomes of the indicated lipid compositions. After ultracentrifugation, the S (liposome-free supernatant) and P (liposome pellet) fractions were analysed by SDS–PAGE and Coomassie blue staining (d). Leakage of the liposomes (e) was monitored in real time by measuring 2,6-pyridinedicarboxylic acid (DPA) chelating-induced fluorescence of released Tb3+. Triton X-100 treatment was used to achieve 100% liposome leakage. f, g, Pyroptotic activity of GSDME-N. cDNAs encoding the indicated GSDME proteins or its caspase-3 cleavage products were transfected into 293T cells (f). Equal amounts of purified GSDME or its caspase-3 cleavage products were electroporated into or added directly to 293T cells (g). Phase-contrast images of cell death morphology are shown in f. ATP cell viability (f, g) is expressed as mean ± s.d. from three technical replicates. Data shown are representative of at least three independent experiments.

Extended Data Figure 4 Characterization of GSDME deafness mutant (GSDMEdeafness) and the switch of chemotherapy drug-induced apoptosis to pyroptosis by caspase-3 cleavage of exogenous GSDME in HeLa cells.

a, b, Pyroptosis-inducing activity of GSDMEdeafness. Full-length human GSDME (GSDME-FL), GSDME-N (the caspase-3 cleavage fragment) or GSDMEdeafness was transiently expressed in 293T cells. a, Phase-contrast images of the cells. b, ATP cell viability expressed as mean ± s.d. from three technical replicates. The immunoblots in b show the expression of transfected GSDME. c, Half life of 3×Flag-tagged full-length GSDME, GSDME-N and GSDMEdeafness in mammalian cells. Twelve hours after transfection of indicated GSDME constructs into 293T cells, 60 μg ml−1 CHX was added to the cells. Total lysates collected at the indicated time points after CHX treatment were subjected to anti-Flag immunoblotting. The triple Flag at the N terminus can inhibit pore-forming activity and thereby inhibit cell death. The upper and lower four panels are from two separate experiments. d, e, Flag-tagged GSDME (wild-type or the D267A or D270A mutant) was stably expressed in GSDMD−/− HeLa cells or GSDMD−/−CASP3−/− HeLa cells. Cells were treated with doxorubucin or 5-FU to induce caspase-3 activation and cell death. d, Phase-contrast images of cell death morphology. e, Anti-Flag and anti-tubulin immunoblotting of total cell lysates. All data shown are representative of at least two independent experiments.

Extended Data Figure 5 Caspase-3 cleavage of GSDME confers pyroptosis in response to chemotherapy drugs in GSDME-positive cancer cells.

a, GSDME expression in various cancer cells. Lysates of indicated cells were blotted with a rabbit monoclonal anti-GSDME antibody and anti-caspase-3 and anti-tubulin antibodies. b, c, Morphological and flow-cytometry assays of chemotherapy drug-induced death in GSDME-negative Jurkat cells and GSDME-positive SH-SY5Y and MeWo cells. b, Phase-contrast cell images. Arrows mark cells showing pyroptotic morphology. c, Jurkat cells were also stained with propidium iodide and annexin V–FITC for flow cytometry analyses. d, Comparison of ATP cell viability and LDH release-based cell death in chemotherapy drug-treated Jurkat cells and effects of zVAD inhibition. ATP cell viability and LDH release are expressed as mean ± s.d. from three technical replicates. e, Immunoblotting assay of chemotherapy drug-induced GSDME cleavage and caspase-3 activation and effects of zVAD. Lysates of Jurkat and MeWo cells treated with indicated chemotherapy drugs were blotted as shown. f, g, Chemotherapy drug-induced pyroptosis is blocked in GSDME−/− SH-SY5Y cells. GSDME−/− SH-SY5Y cells were generated by CRISPR–Cas9-mediated genome editing. f, Top, a related portion of GSDME genomic structure. Bottom, sequences of the targeted region and the two knockout alleles (KO-1 and KO-2). Two guide RNAs (gRNAs) were used to achieve the targeting. Wild-type cells and the two knockout clones were treated with the indicated drugs; LDH release (relative to that of DMSO control)-based cell death shown in g is expressed as mean ± s.d. from three technical replicates. All data shown are representative of three independent experiments.

Extended Data Figure 6 Profiling endogenous GSDME expression in the NCI-60 and effects of decitabine in GSDME-silenced cells.

a, Lysates of 57 cancer cells from the NCI-60 (a panel of 60 diverse human cancer cell lines used by the US National Cancer Institute to screen and evaluate anti-cancer drugs) were subjected to anti-GSDME and anti-tubulin immunoblotting. Wild-type and GSDME−/− SH-SY5Y cells were included as the positive (PC) and negative control (NC), respectively. b, Six indicated GSDME-negative cancer cells were treated with 5 μM decitabine for 6 days and then stimulated with doxorubicin or actinomycin-D for cell death analyses. Top, qRT–PCR measurements of GSDME expression relative to GAPDH before and after decitabine treatment. Middle and bottom, LDH release-based cell death expressed as mean ± s.d. from three technical replicates. All data shown are representative of two independent experiments.

Extended Data Figure 7 Endogenous GSDME in NCI-H522 cells switches chemotherapy drug-induced apoptosis to pyroptosis.

NCI-H522 cells were treated with the indicated chemotherapy drugs. a, Phase-contrast images of cell death morphology. b, LDH release-based cell death expressed as mean ± s.d. from three technical replicates. c, Anti-GSDME immunoblots showing drug-induced cleavage of endogenous GSDME into GSDME-N and GSDME-C. b, c, Effects of zVAD inhibition of caspase activity on cell death and GSDME cleavage. The NCI-H522 cells were also subjected to siRNA knockdown of GSDME expression (knockdown efficiency shown in e) before stimulation with actinomycin-D. d, Cells taken at indicated time points after actinomycin-D stimulation were stained with propidium iodide and annexin V–Alexa 647 for flow cytometry analyses. Rabbit polyclonal anti-GSDME antibody was used in c; rabbit monoclonal antibody capable of recognizing the N-terminal domains of both human and mouse GSDME was used in e. All data shown are representative of two or three independent experiments.

Extended Data Figure 8 Endogenous GSDME in mouse EMT6 cells induces pyroptotic cell death in response to chemotherapy drug treatment.

a, Immunoblotting profiling of GSDME expression in a panel of mouse cancer cells. be, EMT6 cells were treated with actinomycin-D and examined for cell death. b, Cell death determined by measuring LDH release (mean ± s.d.) from three technical replicates. c, Phase-contrast images of cell death morphology. d, Drug-induced cleavage of GSDME and effect of zVAD shown in anti-GSDME immunoblots. e, Effect of zVAD on cell death. A rabbit monoclonal antibody capable of recognizing the N-terminal domains of both human and mouse GSDME was used in a, d. All data shown are representative of three independent experiments.

Extended Data Figure 9 Generation and immune development of Gsdme−/− mice.

a, qRT–PCR analysis of Gsdme transcripts in indicated tissues from wild-type mouse. The mRNA level of Gsdme in mouse melanoma cell line B10-F16 was set as one arbitrary unit. b, Generation of Gsdme−/− mice by CRISPR–Cas9-mediated genome editing. Top, targeted Gsdme locus region. Two gRNAs matching the sequences flanking exon 3 were used to achieve deletion of a large genomic fragment containing exon 3 (193 bp). Bottom, mutated sequences of the knockout allele used for experiments. c, d, Effects of Gsdme−/− on immune cell development in mouse thymus and bone marrow. Total cells in the thymus (c) and bone marrow (d) of wild-type and Gsdme−/− mice were stained with the indicated marker antibodies and subjected to flow cytometry analyses as shown. The flow cytometry plots are from one representative mouse and the statistics of each cell population are expressed as mean ± s.d. (n = 3). Data shown in a, c, d are representative of two independent experiments.

Extended Data Figure 10 GSDME mediates chemotherapy drug-induced tissue damage and toxicity in mice.

ac, Wild-type or Gsdme−/− mice were intraperitoneally injected with cisplatin or saline control. a, Representative haematoxylin and eosin staining of the small intestine (scale bar, 200 μm). White and black arrowheads indicate inflammatory cell infiltration and disappearance of the crypts, respectively. b, P values for the spleen damage data in Fig. 5c. c, Flow-cytometry profiling of the spleen cell population in one representative wild-type and one representative Gsdme−/− mouse. CD3+ and B220+ mark the T and B cell lineages, respectively. dg, Effects of Gsdme knockout on 5-FU-induced small intestine damage and bleomycin-induced lung damage. 5-FU was injected intraperitoneally at a dose of 250 mg per kg per day for 5 days (d, e) and 250 μg bleomycin (per mouse) was delivered intratracheally directly into the lung for 3 days (f, g). d, Left, representative images of the intestine. Right, representative haematoxylin and eosin staining of the small intestine (scale bar, 200 μm). f, Representative haematoxylin and eosin staining of lung sections (scale bar, 20 μm). Statistics for surviving crypts and villi in the small intestine (e) and quantification of lung inflammation and acute lung injury (g) are from 30 randomly selected fields from 3 mice (10 each) and expressed as mean ± s.d. Two-tailed unpaired Student’s t-test was performed in b, e, g (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). All data shown are representative of two independent experiments.

Supplementary information

Supplementary Figure

This file contains the uncropped immunoblots for key data presented in the Main and Extended Data Figures. (PDF 2593 kb)

GSDMA3 can not switch TNFα-induced apoptosis to pyroptosis

Flag-tagged GSDMA3 was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 2.5 h after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). Scale bar, 20 μm. (MP4 5853 kb)

Wild-type GSDME can switch TNFα-induced apoptosis to pyroptosis

Flag-tagged human GSDME was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 30 min after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). Scale bar, 20 μm. (MP4 3997 kb)

GSDME-D270A can not switch TNFα-induced apoptosis to pyroptosis

Flag-tagged human GSDME-D270A mutant was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 2.5 h after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). Scale bar, 20 μm (MP4 7922 kb)

Wild-type GSDME1 can switch TNFα-induced apoptosis to pyroptosis

Flag-tagged wild-type zebrafish GSDME1 was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 30 min after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm. (MP4 9514 kb)

GSDME1-D256A can not switch TNFα-induced apoptosis to pyroptosis

Flag-tagged zebrafish GSDME1-D256A mutant was stably expressed in GSDMD-/- HeLa cells. Shown is the video of a representative field recorded 30 min after stimulation with TNFα + CHX (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm. (MP4 9929 kb)

TNFα-induced apoptosis in control HeLa cells

GSDMD-/- HeLa cells expressing an empty vector, corresponding to the cells assayed in Extended Data Fig. 1f, g, were stimulated with TNFα + CHX. Shown is the video of a representative field recorded 30 min after stimulation (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm. (MP4 9167 kb)

TNFα-induced pyroptosis in GSDMEhigh HeLa cells

GSDMD-/- HeLa cells expressing a high level of GSDME, corresponding to the cells assayed in Extended Data Fig. 1f, g, were stimulated with TNFα + CHX. Shown is the video of a representative field recorded 30 min after stimulation (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm. (MP4 8730 kb)

TNFα-induced secondary pyroptosis after apoptosis in GSDMElow HeLa cells

GSDMD-/- HeLa cells expressing a low level of GSDME, corresponding to the cells assayed in Extended Data Fig. 1f, g, were stimulated with TNFα + CHX. Shown is the video of a representative field recorded 30 min after stimulation (the exact time duration, h : min : s: ms). EGFP in the cytosol was also imaged to highlight the membrane-integrity difference between apoptosis and pyroptosis. Scale bar, 20 μm. (MP4 9369 kb)

Chemotherapy drug-induced apoptosis in GSDME-negative primary HUVEC cells

Cells were treated with Doxorubicin for 2 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm. (MP4 9497 kb)

Chemotherapy drug-induced pyroptosis in GSDME-positive primary NHEK cells

Cells were treated with Doxorubicin for 6 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm. (MP4 9272 kb)

Knockdown of GSDME switches chemotherapy drug-induced pyroptosis to apoptosis in primary NHEK cells

Cells were transfected with GSDME-specific siRNAs. The knockdown cells were treated with doxorubicin for 6 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm. (MP4 9423 kb)

Chemotherapy drug-induced pyroptosis in GSDME-positive primary HPlEpC cells

Cells were treated with doxorubicin for 12 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm. (MP4 7872 kb)

Knockdown of GSDME switches chemotherapy drug-induced pyroptosis to apoptosis in primary HPlEpC cells

Cells were transfected with GSDME-specific siRNAs. The knockdown cells were treated with doxorubicin for 12 h and then recorded for about 6 -7 h (the exact time duration, h : min : s: ms). Shown is the video of a representative field. Scale bar, 20 μm. (MP4 8385 kb)

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Wang, Y., Gao, W., Shi, X. et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547, 99–103 (2017). https://doi.org/10.1038/nature22393

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