Gasdermin E suppresses tumour growth by activating anti-tumour immunity
Authors: Qing Kong
Zhibin Zhang
Xing Liu
Hao Wu
Judy Lieberman
Karla F Meza-Sosa
Ying Zhang
Caroline Junqueira
Temy Mo Yin Mok
Yandan Yao
Satyaki Sengupta
Shunying Li
Shiyu Xia
James Ansara
Journal: Nature (2020)
Discipline: Multidisciplinary
Keywords: Gasdermin E; GSDME; pyroptosis; caspase 3; tumour suppressor
Affiliations: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA. ., Department of Pediatrics, Harvard Medical School, Boston, MA, USA. ., Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA., Department of Pediatrics, Harvard Medical School, Boston, MA, USA., Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA., Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA., Department of Genetics, Harvard Medical School, Boston, MA, USA., Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China., The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China., René Rachou Institute, Oswaldo Cruz Foundation, Belo Horizonte, Brazil., Laboratorio de Neuroimmunobiología, Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico., Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, Hong Kong., Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Abstract:  Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis)1. Gasdermin E (GSDME, also known as DFNA5)-mutated in familial ageing-related hearing loss2-can be cleaved by caspase 3, thereby converting noninflammatory apoptosis to pyroptosis in GSDME-expressing cells3-5. GSDME expression is suppressed in many cancers, and reduced GSDME levels are associated with decreased survival as a result of breast cancer2,6, suggesting that GSDME might be a tumour suppressor. Here we show that 20 of 22 tested cancer-associated GSDME mutations reduce GSDME function. In mice, knocking out Gsdme in GSDME-expressing tumours enhances, whereas ectopic expression in Gsdme-repressed tumours inhibits, tumour growth. This tumour suppression is mediated by killer cytotoxic lymphocytes: it is abrogated in perforin-deficient mice or mice depleted of killer lymphocytes. GSDME expression enhances the phagocytosis of tumour cells by tumour-associated macrophages, as well as the number and functions of tumour-infiltrating natural-killer and CD8+ T lymphocytes. Killer-cell granzyme B also activates caspase-independent pyroptosis in target cells by directly cleaving GSDME at the same site as caspase 3. Uncleavable or pore-defective GSDME proteins are not tumour suppressive. Thus, tumour GSDME acts as a tumour suppressor by activating pyroptosis, enhancing anti-tumour immunity. 

Retracted: No

PubPeer comments:
 The tumor sizes in the first two figures are super small. They are almost under the detection limit by the common measurement. For Fig. 1A, how could the authors measure the tumor size (volume) from 4 to 24 mm^3? Usually tumors smaller than 50 mm^3 are hard for accurate measurement. In Figure 2 and Extended Figure 4J, given the number of tumor cells inoculated as described in the Methods part (1.5X10^5 B16 cells mouse, 2X10^6 CT26 cells mouse), it is hard to believe that the B16 CT26 tumors grow so slowly (Only about 60 mm^3 for B16 tumor at day 18 after inoculation, and only about 12 mm^3 for CT26 tumors at day 10 after inoculation), and it is amazing how the authors measured such small tumors. Probably the only reasonable tumor sizes are in Fig. 2C, B16 tumor size (about 200-300 mm^3 on day 18 after inoculation) on NSG mice. Hope the authors would have an explanation. , 
 The Source Data for Fig. 1a is an Excel spreadsheet that shows the volumes, rather than the raw tumor measurements (e.g. width, length, depth). Many of the volumes are exact integer values, e.g. 6.000 mm^3, 18.000 mm^3, 8.000 mm^3. One explanation is that approximate tumor volume was calculated using the formula 0.5 x length x (width squared), as if pi were used, the volumes would not have been integers. This also suggests tumor dimensions were usually measured to the nearest mm or 0.5 mm. If this is true, it is surprising how consistent the measurements were between the 6 independent tumors, with SEMs rarely greater than 5%. The authors say the measurements were not carried out blind. , 
 Dear colleague: Thank you for bringing this issue up for us to clarify this concern.u00a0 We started tumor size measurement when more than one mouse in the study had a palpable tumor and the largest diameter of the tumor reached around 1 mm. We measured the width and length of each tumor with calipers. Tumor volume is calculated by the modified ellipsoidal formula of 1 2 (lengthxwidth2). A tumor with 1 mm width and length is measurable and has a volume of 0.5 mm3.u00a0 Certainly, measuring these small tumors is not as accurate as measuring a larger tumor, but since tumor growth comparison is relative and we applied the same criteria for measuring each group of mice, the exact size of the tumor will not affect our conclusion that GSDME expression in a tumor inhibits its growth.u00a0 We have also monitored the EV GSDME OE B16 tumor growth for up to 25 days (n=10 mice group)u00a0and the tumor growth curves are attached. As you could see, at later time points the tumors grew faster, especially in the EV group, but GSDME still has a very potent effect in suppressing tumor growth (4 10 mice remained tumor-free for the course of the study).u00a0 The B16 and CT26 cell lines were generous gifts from Dr. Gordon Freeman at the Dana-Farber Cancer Institute. They were transduced with control lentivector (in control group tumors), or lentivirus expressing GSDME or Cas9 guide RNA, which made these tumors more immunogenic, and grew more slowly than wild-type tumors, which is a fact we also noticed. Again, both control and GSDME overexpressing KO tumors were treated with lentivirus or expressed the immunogenic Cas9 protein so the changes in tumor growth due to immunogenicity should be similar. ![file] To comment 2 Dear colleague: 4T1E breast tumors are injected into the mouse mammary fat pad, which better support tumor growth compared to subcutaneous injection (such as is used for B16 tumors, which exhibit larger standard deviations within groups), and usually gave us more uniform tumor growth with lower standard deviations. When comparing tumor growth under the same measurement criteria, we observed significant differences (e.g. between EV and GSDME) as well as no differences (e.g. between EV and GSDME F2A). Thank you! Ying and Zhibin , 
 As the previous peer mentioned, in the Source Data for Fig. 1a, the authors provided the tumor volumes of each mouse from day 6 to day 12. Just look at the tumor volumes of the first mouse in EV group, they are 0.9, 1.27, 1.80, 6.62, 11.88, 15.0, 15.63 mm^3 on each day. By the formula 0.5 X length X (width^2), if we assume the length is the same as width, then the length would be 1.22mm, 1.36mm, 1.53mm, 2.5mm, 2.85mm, 3.11mm, 3.15mm. How could the authors confidently consider these were real tumors when they were so small? I guest most people would worry if the inoculation was successful or not if they have performed the measurement. By the way, EMT6 CT26 B16 are all cell lines commonly used for tumorigenesis, and this is the only paper I ve ever read that recorded so detailed accurate subcutaneous tumor growth when the largest tumor size had barely reached 30 mm^3. ,