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

Anti-GSDME (ab230482) and anti-PCNA antibodies (ab92552) were obtained from Abcam. Anti-ERK (4695), anti-p-ERK (4370), anti-JNK (9252), anti-p-JNK (4668), anti-P38 (8690), and anti-p-P38 (4511) were obtained from Cell Signaling Technology. The neutralizing HMGB1 antibody (ab79823) was purchased from Abcam. Dextran sulfate sodium (DSS) was obtained from MP Biomedicals. AOM (A5486) was purchased from Sigma-Aldrich. Recombinant mouse TNF-α (315-01A) was obtained from PeproTech. Cycloheximide (CHX, C7698), a eukaryote protein synthesis inhibitor, was purchased from Sigma-Aldrich. Recombinant mouse HMGB1 protein (ab181949) was obtained from Abcam. U0126, an ERK1/2 inhibitor, was obtained from InvivoGen. Mouse HMGB1 (E0399m) and IL1a (E0071m) ELISA kits were obtained from EIAab Science Inc, Wuhan. Mouse S100A8 (YXL20093) and S100A9 (YXL20095) ELISA kits were obtained from Yuannuo Science Inc, Chengdu. Mouse LTF (MM-0310M2) and IL33 (MM-0935M2) ELISA kits were obtained from Meimian Science Inc, Guangzhou. Cell counting Kit-8 (CCK8) was obtained from Dojindo Laboratories, Japan.

Endoscopic colonic mucosal biopsy samples were collected from IBD patients and healthy donors at the Nanfang Hospital Gastroenterology Unit. All diagnoses and clinical disease activity assessments were based on a standard combination of clinical, endoscopic and histological assessment and radiologic criteria. All samples were collected from consenting individuals according to the protocols approved by the Ethics Committee of Nanfang Hospital of Southern Medical University. Demographic characteristics are shown in Additional file 1: Table S1.

Gsdme^−/− mice (C57BL/6 strain) were kindly provided by Professor Feng Shao (National Institute of Biological Sciences, Beijing, China). Gsdme^−/− mice and WT mice were bred and maintained in a specific pathogen-free facility, and all animal study protocols were approved by the Institutional Animal Care and Use Committee of Southern Medical University. The Gsdme^−/− and WT mice were littermates and cohoused throughout the experiments. The DSS-induced colitis model was established using a method adapted from a published procedure [18]. Briefly, 8- to 10-week-old male Gsdme^−/− mice and WT littermate controls were administered 2% DSS dissolved in drinking water. The colon tissues of DSS-challenged mice were embedded in paraffin and stained with H&E. Disease activity index scores and inflammation-associated histopathological assessments were performed according to Nature protocols [18]. Intestinal pathology scores were assessed by two pathologists in a double-blind manner.

CAC was induced with AOM/DSS in mice as described elsewhere [19]. Briefly, 8- to 10-week-old male Gsdme^−/− mice and WT littermate controls were intraperitoneally injected with AOM (12 mg/kg). Seven days later, the mice were administered 2% DSS dissolved in drinking water for 7 consecutive days, followed by 14 days of regular drinking water for recovery. This same cycle was repeated twice, subsequently followed by regular drinking water until day 84, when these mice were killed. In the antibody-treated groups, neutralizing HMGB1 antibody was intraperitoneally injected at a dose of 200 µg/mouse on days 1, 3, and 5 during DSS treatment. The colons were collected and cut open longitudinally to measure the tumor numbers and sizes.

IHC was conducted to evaluate the expression levels of GSDME in paraffin-embedded tissues using a specific anti-GSDME antibody. The immunoreactive score (IRS) of each sample was obtained by multiplying the score for the percentage of positive cells (0: no positive cells; 1: < 10% positive cells; 2: 10–50% positive cells; 3: 51–80% positive cells; and 4: > 80% positive cells) and the score for the staining intensity (0: no color reaction; 1: mild reaction; 2: moderate reaction; and 3: intense reaction). All sample IRSs were determined by two independent pathologists who were blinded to both the origin of the samples and the patient outcomes.

Biopsy samples were processed immediately, and IECs were purified using enzymatic digestion as previously described [20]. Briefly, colonic tissues from the WT and KO mice were repeatedly washed in HBSS containing 1 mM DTT and 1% penicillin/streptomycin (Sigma-Aldrich). Then, IECs were isolated by incubation in HBSS (Invitrogen/GIBCO) containing 3 mM EDTA (Sigma-Aldrich). After enzymatic digestion, a 40% Percoll Plus solution (GE Healthcare) was used to remove the mononuclear cells, red blood cells, and dead cells.

Quantitative real-time PCR (qRT-PCR) was performed as previously described [21]. The mRNA levels of target genes were normalized to that of GAPDH. The primers used are shown in Additional file 1: Table S2.

The mouse colon cancer cell line CT26 was obtained from Guangdong Provincial Key Laboratory of Gastroenterology, Southern Medical University. CT26 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (Gibco) in 5% CO₂ at 37 °C.

Cell growth was assessed using the CCK8 assay. Briefly, CT26 cells (1 × 10³ cells/well) were seeded in 96-well plates. The next day, the cells were treated with 1 µg/ml recombinant mouse HMGB1 protein. After 48 h, 10 µl of CCK8 solution was added to each well and incubated for 1 h before the absorbance was measured at 450 nm.

Western blot analysis was performed as previously described [22]. GAPDH was used as an endogenous control. Anti-GSDME, anti-p-p38, total p38, p-JNK, total JNK, p-ERK1/2, total ERK1/2, and anti-PCNA antibodies were diluted 1:1000. Anti-GAPDH was diluted 1:2000. Secondary antibodies were diluted 1:4000. ImageJ software was used to quantify and analyze the density of the protein bands.

For ELISA analysis, IECs from Gsdme^−/− mice and WT littermate controls that were treated with DSS for 7 days were isolated using isolation buffer (5 mM EDTA and 1 mM DTT) and then washed with PBS containing penicillin (100 U/ml) and streptomycin (100 mg/ml) 3 times. Finally, the cells were plated in dishes with DMEM containing 10% FBS for 12 h, and the cell medium was used for ELISA analysis. In our in vitro experiment, isolated IECs from Gsdme^−/− mice and WT littermate controls were treated with TNF-α (50 ng/ml) plus CHX (20 µg/ml) for 12 h, and then, the culture supernatant was collected for ELISA analysis.

The abnormal chronic mucosal inflammation in IBD makes it a refractory intestinal disease [23, 24]. Pyroptosis is a form of proinflammatory cell death [14]. However, it is not yet known whether pyroptosis is involved in the development of mucosal inflammation in IBD. To investigate the involvement of pyroptosis in mucosal inflammation in IBD, we measured the expression levels of gasdermins in the mucosa of IBD patients and found that the protein levels of GSDME were significantly increased in the colonic mucosa of IBD patients compared to healthy controls (Fig. 1a, d). Importantly, the pyroptosis-inducing fragment GSDME-NT could be detected in the inflamed colonic mucosa of IBD patients (Fig. 1b, c) but was not detected in the uninflamed mucosa (Fig. 1c). In addition, we found that GSDME was mainly located in the epithelial cells of the mucosa (Fig. 1d), which was consistent with previous findings showing that gasdermins are mainly expressed in the epithelium of the gastrointestinal tract and skin [25, 26]. Taken together, these results suggest that GSDME-mediated epithelial cell pyroptosis correlates with mucosal inflammation in IBD patients.

To investigate the role of GSDME in colitis development, we established a DSS-induced colitis model with Gsdme^−/− mice and WT littermate controls and found that Gsdme^−/− mice were resistant to DSS-induced colitis, exhibiting less weight loss and disease activity index than WT controls (Fig. 2a, b). Moreover, histological examination revealed that WT mice had severe mucosal inflammation in the colon, while the severity of mucosal inflammation was significantly decreased in Gsdme^−/− mice (Fig. 2d, e). In addition, the levels of GSDME-NT were notably high in the colonic mucosa of WT mice, but GSDME-NT was not detected in the colonic mucosa of Gsdme^−/− mice (Fig. 2c). These results indicate that GSDME-mediated pyroptosis plays an important role in the development of colitis.

Due to the distinctive characteristic of membrane disruption, GSDME-mediated pyroptosis is believed to induce subsequent inflammatory responses through the release of intracellular DAMPs [14]. We investigated why GSDME may facilitate colitis development and proposed a role for DAMPs released from pyroptotic cells in promoting colitis development. Since GSDME was mainly expressed in the intestinal epithelium (Fig. 1d), we measured the intracellular expression levels of DAMPs in IECs and found higher levels of HMGB1 but not other DAMPs in IECs isolated from DSS-challenged WT mice than in IECs from Gsdme^−/− mice (Fig. 3a).

Extracellular HMGB1 can induce inflammatory responses [17]. We next determined the levels of extracellular HMGB1 released from IECs. After DSS exposure, we found higher levels of extracellular HMGB1 released by IECs from WT mice than IECs from Gsdme^−/− mice (Fig. 3b). In addition, the extracellular levels of other kinds of DAMPs were also determined by ELISA. The level of S100A9 but not S100A8, LTF, IL1a, or IL33 released by the IECs from DSS-challenged WT mice was significantly increased compared to that of IECs from Gsdme^−/− mice (Fig. 3c–g).

To further confirm the role of GSDME-mediated pyroptosis in HMGB1 release, we treated isolated IECs with TNF-α and CHX to induce pyroptosis [11]. In the absence of TNF-α and CHX, the levels of extracellular HMGB1 were low and were not significantly different between the WT and Gsdme^−/− groups. After treatment with TNF-α and CHX, the loss of GSDME significantly decreased HMGB1 release (Fig. 3h). In addition, treatment with TNF-α and CHX markedly increased LDH release and GSDME cleavage (Fig. 3i, j) and induced obvious pyroptotic bubbles (Additional file 1: Fig. S1), suggesting that the induction of pyroptosis by TNF-α and CHX was highly efficient. A previous study demonstrated that HMGB1 plays essential roles in the development of DSS-induced colitis [27]. Taken together, these results indicate that GSDME-mediated pyroptosis promotes mucosal inflammation through the release of HMGB1 by IECs.

Patients that have IBD for a long time have a high risk for developing colitis-associated cancer (CAC) [28]. We subsequently investigated whether GSDME participates in the development of CAC. CAC was induced in Gsdme^−/− mice and WT littermate controls with AOM/DSS as previously described [19]. We observed that Gsdme^−/− mice exhibited reduced weight loss and colon shortening and attenuated rectal prolapse (Fig. 4a–d) compared to their WT littermates. Moreover, the numbers and sizes of macroscopically visible tumors from Gsdme^−/− mice were significantly decreased compared to those from their WT littermates (Fig. 4e, f). Importantly, histological examination showed a large number of adenocarcinomas in the mucosa and many malignant cells infiltrating the colonic submucosa of WT mice, but only some adenomas in the mucosa of Gsdme^−/− mice (Additional file 1: Fig. S2).

To explore the role of HMGB1 in CAC tumorigenesis, we first measured HMGB1 expression in mice with AOM/DSS-induced CAC. WT mice exposed to AOM/DSS had obvious GSDME cleavage and increased HMGB1 expression in IECs (Additional file 1: Fig. S3a–c). After GSDME was knocked out, HMGB1 expression was significantly decreased in the colon (Additional file 1: Fig. S3d–g). To further determine the function of HMGB1, we treated AOM/DSS-induced Gsdme^−/− mice and WT littermate controls with a neutralizing anti-HMGB1 antibody. We found obvious decreases in weight loss, colon shortening, rectal prolapse, and tumor numbers and sizes in antibody-treated WT mice compared to untreated WT mice (Fig. 4a–f). In addition, the neutralizing anti-HMGB1 antibody significantly decreased the protein expression of Ki67 and PCNA in the colons of AOM/DSS-challenged WT mice (Additional file 1: Fig. S4a–c). These results indicate that HMGB1 contributes to CAC tumorigenesis, which was consistent with a previous finding showing that a neutralizing anti-HMGB1 antibody dramatically decreased the tumor numbers in DSS-induced Apc^Min/+ mice [27]. However, we also observed that antibody treatment did not significantly decrease the tumor numbers in Gsdme^−/− mice (Fig. 4e). This result suggests that in addition to HMGB1, there could be other tumor-promoting molecules released from cells through GSDME-mediated pyroptosis.

A previous study demonstrated that blocking the RAGE-HMGB1 axis suppresses the growth and metastases of C6 glioma cells by inhibiting the activation of mitogen-activated protein (MAP) kinases, including ERK1/2, p38 and JNK [29]. Hyperactivation of the ERK1/2 pathway, which is composed of the kinases RAS, RAF, MEK, and ERK1/2, drives cancer cell growth in a wide array of cancers [30, 31]. We therefore assessed whether the MAPK pathway contributed to HMGB1-mediated CAC tumorigenesis. We found that the neutralizing anti-HMGB1 antibody significantly decreased ERK1/2 activation (Fig. 5a, b) and PCNA expression (Fig. 6a, b) in AOM/DSS-induced WT mice.

To further investigate the effect of the ERK1/2 pathway on HMGB1-induced cancer cell proliferation, the ERK1/2 inhibitor U0126 was used. In the in vitro experiments, CT26 cells were incubated for 1 h with U0126 before stimulation with HMGB1 for 48 h, and then, cell proliferation was measured by CCK-8 assays. We found that U0126 completely suppressed HMGB1-induced ERK1/2 activation (Fig. 5c, d), cell proliferation and PCNA expression (Fig. 6c–e). Similar results were observed in vivo (Additional file 1: Fig. S5a–e). These results suggest that HMGB1 may participate in CAC tumorigenesis through the ERK1/2 pathway.