HMGB1 released from GSDME-mediated pyroptotic epithelial cells participates in the tumorigenesis of colitis-associated colorectal cancer through the ERK1/2 pathway

(2020) 13:149 Tan et al. J Hematol Oncol https://doi.org/10.1186/s13045-020-00985-0 Open Access RAPID COMMUNICATION HMGB1 released from GSDME‑mediated pyroptotic epithelial cells participates in the tumorigenesis of colitis‑associated colorectal cancer through the ERK1/2 pathway Gao Tan*† , Chongyang Huang†, Jiaye Chen and Fachao Zhi* Abstract Background: Pyroptosis is a form of proinflammatory gasdermin-mediated programmed cell death. Abnormal mucosal inflammation in the intestine is a critical risk factor for colitis-associated colorectal cancer (CAC). However, it is unknown whether pyroptosis participates in the development of CAC. Methods: To investigate the role of gasdermin E (GSDME)-mediated pyroptosis in the development of CAC, Gsdme−/− mice and their wild-type (WT) littermate controls were challenged with azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce a CAC model. Neutralizing antibodies against high-mobility group box protein 1 (HMGB1) were used to determine the role of HMGB1 in CAC. To identify the role of ERK1/2 in HMGB1-induced colon cancer cell proliferation, we performed western blotting and CCK8 assays using the ERK1/2-specific inhibitor U0126 in CT26 colon cancer cells. Results: In the CAC model, Gsdme−/− mice exhibited reduced weight loss and colon shortening, attenuated rectal prolapse, and reduced tumor numbers and sizes compared to WT littermates. Furthermore, treatment with neutralizing anti-HMGB1 antibodies decreased the numbers and sizes of tumors, ERK1/2 activation and proliferating cell nuclear antigen (PCNA) expression in AOM/DSS-challenged WT mice. In addition, our in vitro experiments demonstrated that HMGB1 induced proliferation and PCNA expression in CT26 colon cancer cells through the ERK1/2 pathway. Conclusion: GSDME-mediated pyroptosis promotes the development of CAC by releasing HMGB1, which induces tumor cell proliferation and PCNA expression through the ERK1/2 pathway. This finding reveals a previously unrecognized link between pyroptosis and CAC tumorigenesis and offers new insight into CAC pathogenesis. Introduction Colorectal cancer (CRC) is one of the most common types of fatal malignant tumors worldwide [1]. Recently published data show that CRC is the third leading cause of cancer mortality, accounting for 9% of all *Correspondence: ; † Gao Tan and Chongyang Huang contributed equally to this study Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China cancer-related deaths in the USA [2]. More worryingly, the age at onset is becoming younger age. In individuals less than 50 years old, the incidence and death rate have increased by approximately 2% and 1.3% annually in recent years, respectively [3]. As colitis is one of the predisposing risk factors in CRC, CAC accounts for approximately 5% of CRC cases [4]. Prolonged inflammation is one of the characteristics of tumors, and many cancers develop in response to chronic inflammation or display the hallmarks of prolonged © The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tan et al. J Hematol Oncol (2020) 13:149 inflammation throughout their progression [5–7]. CAC is one of the best examples of tumors that are tightly related to chronic inflammation, which is present in the earliest stage of tumor onset [7]. CAC develops in patients with inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), two clinical phenotypes with risks that are estimated to increase by 0.5–1% per year after 8 to 10 years of IBD [8]. Strikingly, CAC can be delayed or even prevented by treatment with anti-inflammatory drugs, suggesting that inflammatory processes are involved in tumor onset [7]. Pyroptosis was initially considered to be caspase1-mediated necrosis, mainly in response to bacterial invasion [9]. Recent studies have shown that gasdermin D (GSDMD) and GSDME are cleaved by active caspase-1/4/5/11 and caspase-3, respectively, via the middle linker, releasing their gasdermin-N fragments to induce pyroptosis by perforating the cell membrane [10–12]. This pore-forming activity causes cytoplasmic swelling and releases intracellular contents, such as immunogenic damage-associated molecular patterns (DAMPs) [13, 14]. Therefore, pyroptosis has been redefined as gasderminmediated proinflammatory cell death [10, 12]. Because pyroptosis promotes inflammation, it is likely to play an important role in colitis and CAC development. However, it is still not clear whether pyroptosis participates in colitis and CAC development. DAMPs consist of structurally diverse nonpathogenderived molecules, and they share some of the following characteristics: (1) they can bind to and activate cell surface or intracellular pattern recognition receptors (PRRs) [15]; (2) they can be not only actively secreted from stressed cells but can also passively released when the plasma membrane is disrupted following certain forms of cell death, such as necrosis, necroptosis, and pyroptosis [16, 17]; and (3) they may switch from a physiological to a proinflammatory function after being released into the extracellular milieu [17]. Various DAMPs have been recognized, including HMGB1, lactoferrin (LTF), S100 proteins A8 and A9 (S100A8/9), IL1a, and IL33 [17]. However, the functional relevance and the effects of these DAMPs on CAC are not entirely clear. The purpose of this study was to determine the role of gasdermin-mediated pyroptosis in colitis and CAC development. For this purpose, we explored the significance of gasdermin-mediated pyroptosis in experimentally induced colitis and CAC and elucidated its important role in colitis and CAC pathogenesis. Methods Antibodies and reagents Anti-GSDME (ab230482) and anti-PCNA antibodies (ab92552) were obtained from Abcam. Anti-ERK Page 2 of 11 (4695), anti-p-ERK (4370), anti-JNK (9252), anti-p-JNK (4668), anti-P (...truncated)