June 15, 2018 (Vol. 38, No. 12)

Chemotoxicity Reduction Efforts May Focus on GSDME, an Apoptosis-to-Pyroptosis Switch

Chemotherapy administered to cancer patients has a variety of adverse side effects such as tissue damage, reduced immunity, and weight loss. Preventing these unwanted side effects could improve the quality of life for patients and potentially increase treatment success.

Attempts to understand chemotherapy toxicity extend to gasdermin, a family of proteins normally found in epithelial, hematopoietic, and many other tissues that have a role in immune defense. Some gasdermin family members, including the poorly understood gasdermin E (GSDME), have been found to be directly involved in cancer and tumor suppression.

According to recent research, GSDME has an activated form that can induce pyroptosis, an inherently inflammatory form of cell death. GSDME, then, has implications for cancer treatment and diagnosis.1

GSDME expression levels in normal cells play a role in chemotherapy’s toxic side effects. In GSDME-expressing cells, chemotherapy drugs or inflammatory factors that normally activate apoptosis-mediated cell death may switch to the activation of pyroptosis, an alternative cell-death pathway that contributes to the cytotoxicity of chemotherapy drugs in normal tissues.

This apoptosis-to-pyroptosis switch raises two interesting questions:

  • Can inhibiting or reducing the expression of GSDME decrease chemotherapy-related side effects?
  • Could the detection of GSDME expression be used in the diagnosis or prognosis of cancer?

To answer such questions, the development of suitable and reproducible detection tools is essential.

A New Look at Programmed Cell Death

For a long time, pyroptosis has been regarded simply as caspase-1-mediated monocyte death, mostly in response to bacterial insult. We now know that pyroptosis is actually activated by the canonical caspase-1 “inflammasomes” as well as by the activation of caspase-4, -5, and -11 by cytosolic lipopolysaccharide, in which case it functions as a general innate immune mechanism.

In these contexts, gasdermin D (GSDMD) serves as the pyroptosis “executioner” protein, due to its cleavage and activation by caspase-1, -4, -5, and -11. In addition, a recent study showed that GSDME can be cleaved and activated by caspase-3 to cause pyroptosis. Together, these mechanistic insights redefine pyroptosis as gasdermin-mediated programmed necrosis. They have changed our fundamental understanding of programmed cell death.

Animal and cellular models that express reduced levels of GSDME were used to investigate the possibility of alleviating or even avoiding GSDME-mediated pyroptosis and its inflammatory effects. To carry out this research and examine the role of GSDME in chemotherapy, researchers at National Institute of Biological Sciences in collaboration with scientists at Abcam developed antibodies to GSDME.

Specifically, these investigators developed highly sensitive and specific recombinant rabbit monoclonal antibodies, generated via RabMAb® technology, that recognize the N-terminal domain of human and mouse GSDME. The investigators also developed a rabbit monoclonal antibody to the C-terminal domain of human GSDME.

It was important to be able to differentiate GSDME N- and C-terminal domains as caspase-3 cleavage of GSDME generates a GSDME-N fragment, which is the cleaved protein responsible for membrane perforation and the induction of pyroptosis. These antibodies gave the team the ability to accurately profile GSDME expression levels in both cancerous and endogenous cells and tissues.

The work with animal and cellular models confirmed that chemotherapy drugs trigger pyroptosis via GSDME, and that this process was partly responsible for the adverse side effects that patients experience when on chemotherapy. This is because GSDME expression can switch chemotherapy-induced apoptosis to pyroptosis via the pore-forming activity of caspase-3-cleaved GSDME.2 This form of GSDME-mediated pyroptosis is central to eliciting extensive inflammatory damage and the toxic side effects witnessed in conventional chemotherapy.

Gasdermin and Cancer

Both tumors and normal tissues can express GSDME at varying levels. The recombinant rabbit monoclonal GSDME antibodies generated during this project were capable of detecting GSDME at very low levels and helped characterize GSDME expression across a range of cell types, including normal noncancer cells and NCI-60, which is a panel of 60 diverse human cancer cell lines used by the U.S. National Cancer Institute to screen and evaluate anticancer drugs.

Although most of the cancer cells showed silenced expression of GSDME, the team identified a few human cell lines, including SH-SY5Y neuroblastoma and MeWo skin melanoma cells, that expressed GSDME at high levels. With these two cell lines, the team was able to demonstrate that the switch to the GSDME-mediated pyroptotic pathway occurred in response to different agents, such as tumor necrosis factor or chemotherapy drugs, and/or forms of DNA damage known to cause caspase-3 activation.

This work showed that caspase-3 cleaves and activates GSDME to cause pyroptosis, and that it is the expression level of GSDME that determines the type of programmed cell death that occurs in caspase-3-activated cells. Cells with high expression levels of GSDME underwent pyroptosis upon treatment with a chemotherapy drug that normally causes apoptotic stimulation.

Moreover, in SH-SY5Y neuroblastoma and MeWo cell lines, chemotherapy drug–induced pyroptosis was inhibited in the presence of zVAD, a caspase inhibitor that prevents cleavage of GSDME. When a human knockout cell line was used, GSDME−/− SH-SY5Y, no chemotherapy drug-induced pyroptosis occurred. Meanwhile, cells with low or absent GSDME levels were found to develop secondary necrosis (pyroptosis) following apoptosis activation.3

During tumorigenesis, GSDME undergoes epigenetic silencing by methylation. This is why most cancer cells do not express GSDME. This raises the interesting possibility that reversing the epigenetic silencing may sensitize these cells to chemotherapy agents.

In a test of this possibility, cells that had lost GSDME expression were treated with azacitidine, a cancer drug that is approved for treating myelodysplastic syndromes, myeloid leukemia, or chronic myelomonocytic leukemia. Azacitidine inhibited methyltransferases, reversed the methylation/silencing of GSDME, and increased the expression of GSDME. Consequently, when additional chemotherapeutic drugs were administered, induction of the pyroptotic pathway diminished.

As opposed to cancer cells, many normal cells do express GSDME at high levels. Thus, chemotherapy agents sensitize normal cells and force them down the pyroptotic pathway. One reason conventional chemotherapy drugs cause such severe cytotoxicity is the large degree of inflammatory damage caused by pyroptosis in normal tissues.

To test whether GSDME contributes to the adverse effects of chemotherapy drugs in a whole animal model, investigators exposed knockout Gsdme−/− mice to cisplatin (a chemotherapy drug) and examined the small intestine, spleen, lung, and other tissues. This work generated data supporting the hypothesis that GSDME-mediated pyroptosis results in inflammatory damage. GSDME, then, could play a major role in chemotherapy drug-induced cytotoxicity. If so, GSDME expression modulation could be an effective means of managing inflammation and thus the adverse side effects of chemotherapy.

What’s Next?

Research is now being directed toward understanding whether the pyroptosis pathway is mediated by GSDMD and/or GSDME, and whether the downstream inflammatory response contributes to tumorigenesis or plays a role in modulating the tumor immune response. It is unclear whether pyroptosis-targeted immunotherapy could improve cancer treatment. To resolve this question, new studies are needed. They could examine whether pyroptosis regulates the killing of tumor cells via cytokine induction. Also, they could determine whether pyroptosis could modulate effectiveness of checkpoint-blockade mediators in cancerous immune cells.

The research conducted thus far has changed our understanding of pyroptosis and offered new insights into programmed cell death. In addition, it has shed light on the possibility that numerous side effects of chemotherapy could be circumvented by modulating GSDME expression. A clear link between GSDME expression levels and the type of cell death that occurs also means that further profiling of GSDME levels could result in new diagnostic and prognostic antibody-based tools for cancer.

Feng Shao, Ph.D. ([email protected]) is investigator and deputy director for academic affairs at the National Institute of Biological Sciences, Beijing
1. Wang, Y. et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 2017; 547: 99–103.
2. Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 2016; 535: 111–116.
3. Rogers, C. et al. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. 2017; Nat. Commun. 8: 1–14.
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