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May 1, 2017 -Studies from Dr. Feng Shao’s laboratory discover that caspase-3 can cleave GSDME to trigger pyroptosis which contributes to the toxic effect of chemotherapy.

Publication Date:2017/05/09

May 1, 2017 -Studies from Dr. Feng Shao’s laboratory discover that caspase-3 can cleave GSDME to trigger pyroptosis which contributes to the toxic effect of chemotherapy. The work entitled “Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a Gasdermin” is published in the journal Nature as Accelerated Article Preview.

Pyroptosis traditionally refers to caspase-1-mediated programmed and proinflammatory death in monocytes, but its nature and mechanism was unknown. Previous work from Dr. Shao’s laboratory reveals that caspase-4/5/11, as the cytosolic receptors for bacterial LPS, can also trigger pyroptosis even in non-monocytic cells, which determines LPS-induced sepsis (Shi et al., Nature 2014). In the past two years, the Shao laboratory has identified a novel Gasdermin-D (GSDMD) protein, a common substrate of caspase-1 and caspase-4/5/11. GSDMD is normally kept in an autoinibited state and upon caspase cleavage then releases its N-terminal domain that perforates the membrane and causes cell swelling and eventual lysis (Shi et al., Nature 2015;Ding et al., Nature 2016). These findings establish that GSDMD is the executioner of caspase-1/4/5/11-induced pyroptosis and clarify that pyroptosis is indeed necrotic. More importantly, GSDMD represents a large Gasdermin family that also includes GSDMA, GSDMB, GSDMC, DFNA5/GSDME and DFNB59. As the Gasdermin family shares an N-terminal pore-forming and pro-pyroptotic domain (Ding et al., Nature 2016), the Shao team recently redefines the concept of pyroptosis as Gasdermin-mediated programmed necrotic cell death (Shi et al., TiBS 2017).

Previously, the Shao team found that replacing the caspase-1/11 cleaving site in GSDMD with the caspase-3 site could switch TNFa-induced caspase-3-mediated HeLa cell apoptosis to pyroptosis. In subsequent control assays, they unexpectedly observed that expression of wild-type GSDME caused a similar apoptosis-to-pyroptosis switch. Sequence analyses then identified a caspase-3-cleavage site between the N- and C-terminal domains in GSDME. Indeed GSDME was cleaved upon TNFa stimulation; mutation of the cleavage site not only blocked caspase-3 cleavage but also reversed cell pyroptosis to apoptosis. The authors then performed a series of in vitro biochemical analyses, and found that purified GSDME was specifically cleaved by caspase-3 at the expected site with the cleavage efficiency even higher than that of a known caspase-3 substrate RhoGDI. Like GSDMD cleavage by caspase-1, the N-terminal fragment of GSDME after caspase-3 cleavage could bind to PI(4,5)P2 in the liposome and render leakage of the liposome; electron microscopy recorded many pores of regular shape and size on the liposome. These results establish that GSDME is cleaved and activated by caspase-3, which then perforate the membrane to cause pyroptosis.

As most commonly used cell lines lack GSDME expression, the researchers were only able to identify human neuroblastoma SH-SY5Y and skin melanoma MeWo cells that had high endogenous GSDME expression. When the two cells were treated with DNA-damaging chemotherapy drugs, caspase-3 activation occurred and the cells died out of pyroptosis. In contrast and also consistent with the prevailing notion, Jurkat cells that expressed no GSDME underwent classical apoptosis following the drug treatment. Subsequent experiments confirmed that chemotherapy drugs-induced pyroptosis in SH-SY5Y cells was caused by caspase-3 cleavage of GSDME. The Shao team further profiled 57 different cancer cells in the NCI-60 and found that only 1/10 of them had decent GSDME expression. This agrees with previous literatures showing that GSDME is silenced in cancer due to promoter methylation. The GSDME-positive cells, like the non-small cell lung carcinoma NCI-H522, behaved similarly as SH-SY5Y cells and developed pyroptosis in a GSDME-dependent manner upon chemotherapy drug treatment. Meanwhile, the GSDME-negative cancer cells became more sensitive to chemotherapy drugs after reversing GSDME silencing using the DNA methyltransferase inhibitor decitabine, an FDA-approved anticancer drug.

Different from cancer cells, many normal cells and tissues do express a high-level GSDME. The researchers then checked five primary cells from various normal human tissues and found three of them positive for GSDME expression. When the primary cells were treated various chemotherapy drugs, the positive cells developed pyroptosis and showed caspase-3-dependent GSDME cleavage while the two GSDME-negative cells underwent apoptosis. When GSDME expression was knocked down, chemotherapy drugs-induced death in the positive cells were converted from pyroptosis to apoptosis. Given that apoptosis, as a “clean” cell death, could not account for the severe toxicity observed with chemotherapy drugs in clinics, the researchers hypothesized that GSDME-mediated pyroptosis might be important for the chemotoxicity. To test the idea, they generated Gsdme deficient mice. When mice were treated with Cisplatin, severe damages in the small intestine, spleen and lung as well as strong inflammation in the intestine and lung were observed in wild-type mice; concomitantly, weight loss of the mice was apparent. Importantly, all these toxic effects were evidently alleviated in Gsdme deficient mice. This finding was further confirmed in 5-FU-induced small intestine damage and belomycin-induced lung inflammatory damage.

The study for the first time shows that pyroptosis mediated by the Gasdermin-family member GSDME is the key for the toxic effect of conventional chemotherapy drugs, providing new insights into cancer therapy. This is also the first time to show that pyroptosis has important functions outside of innate immunity. The study brings a new concept that caspase-3 can also trigger necrosis (pyroptosis), breaking the dogma that caspase-3 is a hallmark of apoptosis.

PhD students Yupeng Wang and Wenqing Gao from the Shao laboratory are co-first authors of the study; student Xuyan Shi also made significant contribution; other contributors include Dr. Jingjin Ding, Wang Liu, Huabin He and Kun Wang from the Shao laboratory. Dr. Feng Shao is the corresponding author of the paper. The research was supported by the National Key Research and Development Project, the Beijing Municipal Government, the Strategic Priority Research Program of the Chinese Academy of Sciences and Howard Hughes Medical Institute in the States, and carried out at National Institute of Biological Sciences, Beijing.