Genetic Engineering & Biotechnology News

JUN15 2018

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Page 20 of 37 | Genetic Engineering & Biotechnology News | JUNE 15, 2018 | 19 Feng Shao, Ph.D. Chemotherapy administered to cancer patients has a variety of adverse side effects such as tis- sue 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 tox- icity extend to gasdermin, a family of proteins normally found in epithelial, hematopoietic, and many other tissues that have a role in im- mune defense. Some gasdermin family mem- bers, including the poorly understood gasder- min E (GSDME), have been found to be direct- ly involved in cancer and tumor suppression. According to recent research, GSDME has an activated form that can induce py- roptosis, 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 ef- fects. In GSDME-expressing cells, chemo- therapy drugs or inflammatory factors that normally activate apoptosis-mediated cell death may switch to the activation of pyrop- tosis, an alternative cell-death pathway that contributes to the cytotoxicity of chemother- apy drugs in normal tissues. This apoptosis-to-pyroptosis switch rais- es two interesting questions: 1. Can inhibiting or reducing the expression of GSDME decrease chemotherapy-related side effects? 2. Could the detection of GSDME expression be used in the diagnosis or prognosis of cancer? To answer such questions, the develop- ment of suitable and reproducible detection tools is essential. A New Look at Programmed Cell Death For a long time, pyroptosis has been re- garded simply as caspase-1-mediated mono- cyte death, mostly in response to bacterial insult. We now know that pyroptosis is actu- ally activated by the canonical caspase-1 "in- flammasomes" as well as by the activation of caspase-4, -5, and -11 by cytosolic lipo- polysaccharide, in which case it functions as a general innate immune mechanism. In these contexts, gasdermin D (GSDMD) serves as the pyroptosis "executioner" pro- tein, due to its cleavage and activation by cas- pase-1, -4, -5, and -11. In addition, a recent study showed that GSDME can be cleaved and activated by caspase-3 to cause pyropto- sis. Together, these mechanistic insights rede- fine pyroptosis as gasdermin-mediated pro- grammed necrosis. They have changed our fundamental understanding of programmed cell death. Animal and cellular models that express reduced levels of GSDME were used to in- vestigate the possibility of alleviating or even avoiding GSDME-mediated pyroptosis and its inflammatory effects. To carry out this re- search and examine the role of GSDME in chemotherapy, researchers at National Insti- tute of Biological Sciences in collaboration with scientists at Abcam developed antibod- ies 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 GSD- ME. The investigators also developed a rab- bit 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 cas- pase-3 cleavage of GSDME generates a GSD- ME-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 pro- cess was partly responsible for the adverse side effects that patients experience when on chemotherapy. This is because GSDME ex- pression 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 cen- tral to eliciting extensive inflammatory dam- age and the toxic side effects witnessed in conventional chemotherapy. Gasdermin and Cancer Both tumors and normal tissues can ex- press GSDME at varying levels. The recombi- nant 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 iden- tified a few human cell lines, including SH- SY5Y neuroblastoma and MeWo skin mela- noma cells, that expressed GSDME at high levels. With these two cell lines, the team was able to demonstrate that the switch to the GS- DME-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 cas- pase-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 hu- man 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) fol- lowing apoptosis activation. 3 During tumorigenesis, GSDME under- goes epigenetic silencing by methylation. This is why most cancer cells do not express GSDME. This raises the interesting possibili- ty 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 leuke- mia. Azacitidine inhibited methyltransferases, reversed the methylation/silencing of GSDME, and increased the expression of GSDME. Consequently, when additional chemothera- peutic 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 path- way. One reason conventional chemothera- py 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 che- motherapy drug) and examined the small in- testine, spleen, lung, and other tissues. This work generated data supporting the hypoth- esis that GSDME-mediated pyroptosis results in inflammatory damage. GSDME, then, could play a major role in chemotherapy drug- induced cytotoxicity. If so, GSDME expres- sion modulation could be an effective means of managing inflammation and thus the ad- verse side effects of chemotherapy. What's Next? Research is now being directed toward understanding whether the pyroptosis path- way is mediated by GSDMD and/or GSD- ME, and whether the downstream inflamma- tory response contributes to tumorigenesis or plays a role in modulating the tumor immune response. It is unclear whether pyroptosis-tar- geted immunotherapy could improve cancer treatment. To resolve this question, new stud- ies are needed. They could examine whether pyroptosis regulates the killing of tumor cells via cytokine induction. Also, they could de- termine whether pyroptosis could modulate effectiveness of checkpoint-blockade media- tors in cancerous immune cells. The research conducted thus far has changed our understanding of pyroptosis and offered new insights into programmed cell death. In ad- dition, it has shed light on the possibility that numerous side effects of chemotherapy could be circumvented by modulating GSDME ex- pression. A clear link between GSDME expres- sion levels and the type of cell death that occurs also means that further profiling of GSDME levels could result in new diagnostic and prog- nostic antibody-based tools for cancer. References available online. Feng Shao, Ph.D. ( is investigator and deputy director for academic affairs at the National Institute of Biological Sciences, Beijing. Website: Developing Detection Tools for Gasdermin E Tutorial OMICS Chemotoxicity Reduction Efforts May Focus on GSDME, an Apoptosis-to-Pyroptosis Switch Gasdermin E (GSDME) expression levels control the mode of programmed cell death in response to chemotherapy drugs.

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