Caspase-4 in glioma indicates deterioration and unfavorable prognosis by affecting tumor cell proliferation and immune cell recruitment.

Gliomas are the most common malignant tumors of the central nervous system, accounting for approximately 80% of all malignant brain tumors. Accumulating evidence suggest that pyroptosis plays an essential role in the progression of cancer. Unfortunately, the effect of the pyroptosis-related factor caspase-4 (CASP4) on immunotherapy and drug therapy for tumors has not been comprehensively investigated. In this study, we systematically screened six hub genes by pooling differential pyroptosis-related genes in The Cancer Genome Atlas (TCGA) glioma data and the degree of centrality of index-related genes in the protein-protein interaction network. We performed functional and pathway enrichment analyses of the six hub genes to explore their biological functions and potential molecular mechanisms. We then investigated the importance of CASP4 using Kaplan-Meier survival analysis of glioma patients. TCGA and the Chinese Glioma Genome Atlas (CGGA) databases showed that reduced CASP4 expression leads to the potent clinical deterioration of glioma patients. Computational analysis of the effect of CASP4 on the infiltration level and recruitment of glioma immune cells revealed that CASP4 expression was closely associated with a series of tumor-suppressive immune checkpoint molecules, chemokines, and chemokine receptors. We also found that aberrant CASP4 expression correlated with chemotherapeutic drug sensitivity. Finally, analysis at the cellular and tissue levels indicated an increase in CASP4 expression in glioma, and that CASP4 inhibition significantly inhibited the proliferation of glioma cells. Thus, CASP4 is implicated as a new prognostic biomarker for gliomas with the potential to further guide immunotherapy and chemotherapy strategies for glioma patients.

The non-canonical inflammasome activators Caspase-4 and Caspase-5 are differentially regulated during immunosuppression-associated organ damage.

The non-canonical inflammasome, which includes caspase-11 in mice and caspase-4 and caspase-5 in humans, is upregulated during inflammatory processes and activated in response to bacterial infections to carry out pyroptosis. Inadequate activity of the inflammasome has been associated with states of immunosuppression and immunopathological organ damage. However, the regulation of the receptors caspase-4 and caspase-5 during severe states of immunosuppression is largely not understood. We report that CASP4 and CASP5 are differentially regulated during acute-on-chronic liver failure and sepsis-associated immunosuppression, suggesting non-redundant functions in the inflammasome response to infection. While CASP5 remained upregulated and cleaved p20-GSDMD could be detected in sera from critically ill patients, CASP4 was downregulated in critically ill patients who exhibited features of immunosuppression and organ failure. Mechanistically, downregulation of CASP4 correlated with decreased gasdermin D levels and impaired interferon signaling, as reflected by decreased activity of the CASP4 transcriptional activators IRF1 and IRF2. Caspase-4 gene and protein expression inversely correlated with markers of organ dysfunction, including MELD and SOFA scores, and with GSDMD activity, illustrating the association of CASP4 levels with disease severity. Our results document the selective downregulation of the non-canonical inflammasome activator caspase-4 in the context of sepsis-associated immunosuppression and organ damage and provide new insights for the development of biomarkers or novel immunomodulatory therapies for the treatment of severe infections.

Identification of a novel pyroptosis-related gene signature in human spermatogenic dysfunction.

PURPOSE: To reveal the underlying roles that pyroptosis-related genes (PRGs) played in human spermatogenic dysfunction. METHODS: One discovery set and three validation sets were employed to inspect the previously reported 33 PRGs in the human testis with different status of spermatogenesis. PRGs that differentially expressed in all sets were considered as key differentially expressed pyroptosis-related genes (PR-DEGs). The relationships between key PR-DEGs and samples' clinicopathological, therapeutic, and immune patterns were respectively studied. Single-cell RNA sequencing (scRNS-seq) analyses were conducted to show the expression changes and related mechanisms of key PR-DEGs at a single-cell resolution. RESULTS: CASP4 and GPX4 were identified as two key PR-DEGs. These two genes were significantly dysregulated in spermatogenic dysfunctional samples, but with opposite tendency. CASP4 was negatively correlated with Johnsen scores but positively correlated with follicle-stimulating hormone (FSH) levels (all p < 0.05), while GPX4 exhibited significant positive correlations with Johnsen scores and negative relevance with FSH. For treatments, both molecules showed a prospective value of being predictors for sperm retrieval surgeries. Moreover, CASP4 and GPX4 were potential immunoregulators in the testicular immune microenvironment and showed significant correlations to testicular macrophages and mast cell infiltration. In scRNA-seq analyses, GPX4 was highly expressed in germ cells, which therefore suffered a sharp reduction with the loss of germ cells in spermatogenic dysfunction. On the other hand, CASP4 were basically somatic cell-derived, and the proportion of CASP4-positive Leydig cells significantly increased in disease testes (p = 0.0001). CONCLUSION: In all, we revealed two key PRGs of human testes that might be functional in spermatogenic dysfunction.

Testis cell pyroptosis mediated by CASP1 and CASP4: possible sertoli cell-only syndrome pathogenesis.

BACKGROUND: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. Recently, several genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot fully explain the pathogenesis of SCOS. This study attempted to explain spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and to provide new targets for SCOS diagnosis and therapy. METHODS: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry. RESULTS: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 ß, IL-18, LDH, and ROS) were significantly increased in the SCOS group. CONCLUSIONS: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.

Inhibition of IL1R1 or CASP4 attenuates spinal cord injury through ameliorating NLRP3 inflammasome-induced pyroptosis.

Spinal cord injury (SCI) is a devastating trauma characterized by serious neuroinflammation and permanent neurological dysfunction. However, the molecular mechanism of SCI remains unclear, and few effective medical therapies are available at present. In this study, multiple bioinformatics methods were used to screen out novel targets for SCI, and the mechanism of these candidates during the progression of neuroinflammation as well as the therapeutic effects were both verified in a rat model of traumatic SCI. As a result, CASP4, IGSF6 and IL1R1 were identified as the potential diagnostic and therapeutic targets for SCI by computational analysis, which were enriched in NF-κB and IL6-JAK-STATA3 signaling pathways. In the injured spinal cord, these three signatures were up-regulated and closely correlated with NLRP3 inflammasome formation and gasdermin D (GSDMD) -induced pyroptosis. Intrathecal injection of inhibitors of IL1R1 or CASP4 improved the functional recovery of SCI rats and decreased the expression of these targets and inflammasome component proteins, such as NLRP3 and GSDMD. This treatment also inhibited the pp65 activation into the nucleus and apoptosis progression. In conclusion, our findings of the three targets shed new light on the pathogenesis of SCI, and the use of immunosuppressive agents targeting these proteins exerted anti-inflammatory effects against spinal cord inflammation by inhibiting NF-kB and NLRP3 inflammasome activation, thus blocking GSDMD -induced pyroptosis and immune activation.

Salmonella enterica Serovar Typhimurium Induces NAIP/NLRC4- and NLRP3/ASC-Independent, Caspase-4-Dependent Inflammasome Activation in Human Intestinal Epithelial Cells.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes diseases ranging from gastroenteritis to systemic infection and sepsis. Salmonella uses type III secretion systems (T3SS) to inject effectors into host cells. While these effectors are necessary for bacterial invasion and intracellular survival, intracellular delivery of T3SS products also enables detection of translocated Salmonella ligands by cytosolic immune sensors. Some of these sensors form multimeric complexes called inflammasomes, which activate caspases that lead to interleukin-1 (IL-1) family cytokine release and pyroptosis. In particular, the Salmonella T3SS needle, inner rod, and flagellin proteins activate the NAIP/NLRC4 inflammasome in murine intestinal epithelial cells (IECs), which leads to restriction of bacterial replication and extrusion of infected IECs into the intestinal lumen, thereby preventing systemic dissemination of Salmonella. While these processes are quite well studied in mice, the role of the NAIP/NLRC4 inflammasome in human IECs remains unknown. Unexpectedly, we found the NAIP/NLRC4 inflammasome is dispensable for early inflammasome responses to Salmonella in both human IEC lines and enteroids. Additionally, NLRP3 and the adaptor protein ASC are not required for inflammasome activation in Caco-2 cells. Instead, we observed a necessity for caspase-4 and gasdermin D pore-forming activity in mediating inflammasome responses to Salmonella in Caco-2 cells. These findings suggest that unlike murine IECs, human IECs do not rely on NAIP/NLRC4 or NLRP3/ASC inflammasomes and instead primarily use caspase-4 to mediate inflammasome responses to Salmonella pathogenicity island 1 (SPI-1)-expressing Salmonella.

Caspase-4/11 is critical for angiogenesis by repressing Notch1 signalling via inhibiting γ-secretase activity.

BACKGROUND AND PURPOSE: Notch1 activation mediated by γ-secretase is critical for angiogenesis. GeneCards database predicted that Caspase-4 (CASP4, with murine ortholog CASP11) interacts with presenilin-1, the catalytic core of γ-secretase. Therefore, we investigated the role of CASP4/11 in angiogenesis. EXPERIMENTAL APPROACH: In vivo, we studied the role of Casp11 in several angiogenesis mouse models using Casp11 wild-type and knockout mice. In vitro, we detected the effects of CASP4 on endothelial functions and Notch signalling by depleting or overexpressing CASP4 in human umbilical vein endothelial cells (HUVECs). The functional domain responsible for the binding of CASP4 and presenilin-1 was detected by mutagenesis and co-immunoprecipitation. KEY RESULTS: Casp11 deficiency impaired adult angiogenesis in ischaemic hindlimbs, melanoma xenografts and Matrigel plugs, but not the developmental angiogenesis of retina. Bone marrow transplantation revealed that the pro-angiogenic effect depended on CASP11 derived from non-haematopoietic cells. CASP4 expression was induced by inflammatory factors and CASP4 knockdown decreased cell viability, proliferation, migration and tube formation in HUVECs. Mechanistically, CASP4/11 deficiency increased Notch1 activation in vivo and in vitro, while CASP4 overexpression repressed Notch1 signalling in HUVECs. Moreover, CASP4 knockdown increased γ-secretase activity. The γ-Secretase inhibitor DAPT restored the effects of CASP4 siRNA on Notch1 activation and angiogenesis in HUVECs. Notably, the catalytic activity of CASP4/11 was dispensable. CASP4 directly interacted with presenilin-1 through the caspase recruitment domain (CARD). CONCLUSIONS AND IMPLICATIONS: These findings reveal a critical role of CASP4/11 in adult angiogenesis and make this molecule a promising therapeutic target for angiogenesis-related diseases in the future.

Pyroptosis-related genes regulate proliferation and invasion of pancreatic cancer and serve as the prognostic signature for modeling patient survival.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has high mortality and poor prognosis. Pyroptosis can influence the prognosis of patients by regulating the proliferation, invasion, and metastasis of cancer cells. However, the role of pyroptosis-related genes (PRGs) in PDAC remains unclear. METHODS: In this study, based on the Cancer Genome Atlas (TCGA) cohort of PDAC samples, univariate Cox analysis and LASSO regression analysis were used to screen the prognostic PRGs and establish the gene signature. To further evaluate the functional significance of CASP4 and NLRP1 in PDAC, we also conducted an in vitro study to explore the mechanism of CASP4 and NLRP1 regulating the occurrence and development of PDAC. Finally, we investigated the relationship between CASP4 and NLRP1 expression levels and drug sensitivity in pancreatic cancer cells. RESULTS: A risk prediction model based on CASP4 and NLRP1 was established, which can distinguish high-risk patients from low-risk patients (P < 0.001). Both internal validation and external GEO data sets validation demonstrate good predictive capability of the model (AUC = 0.732, AUC = 0.802, AUC = 0.632, P < 0.05). In vitro, CCK8 and Transwell assay suggested that CASP4 may accelerate the progression of PDAC by promoting proliferation and migration of pancreatic cancer cells, while NLRP1 has been found to have tumor suppressive effect. It should be noted that knockdown of CASP4 reduced the level of coke death, the expression levels of acetyl-CoA carboxylase, FASN, SREBP-1 and SREBP-2 were decreased, and the number of lipid droplets was also significantly reduced. Moreover, the enrichment of signaling pathways showed that NLRP1 was significantly correlated with MAPK and RAS/ERK signaling pathways, and knocking down NLRP1 could indeed up-regulate p-ERK expression. Finally, high expression of CASP4 and low expression of NLRP1 increased the sensitivity of pancreatic cancer cells to ERK inhibitors. CONCLUSIONS: In especial, CASP4 can promote tumor progression by promoting the synthesis and accumulation of fatty acids, while NLRP1 acts on RAS/ERK signaling pathway. Both of genes play an important role in the diagnosis and treatment of PDAC, which may also affect the inhibitors of MAPK/ERK efficiency.

Identification of a Pyroptosis-Related Gene Signature and Effect of Silencing the CHMP4C and CASP4 in Pancreatic Adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor with an extremely poor prognosis. Pyroptosis has been demonstrated to play an important role in tumor prognosis. However, the expression of pyroptosis-related genes in PAAD and their correlations with prognosis remains unclear. Methods: In this study, the 36 pyroptosis-related genes that were differentially expressed between normal pancreatic tissues and PAAD tissues were identified via the "limma" R package. Based on these differentially expressed genes (DEGs), a five-gene signature was established by applying the least absolute shrinkage and selection operator Cox regression in the TCGA cohort and was validated in the GEO cohort. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs based on the risk model indicated that immune-associated biological processes and pathways were enriched. In vivo, we detected the expressions of CASP4 and CHMP4C by immunohistochemistry in tumor tissues and adjacent normal tissues. In vitro, we silenced CASP4 and CHMP4C to explore their effects on pancreatic cancer cells. Results: PAAD patients in the low-risk group showed significantly higher survival possibilities than those in the high-risk group. The expressions of CASP4 and CHMP4C in tumor tissue were higher than those in the adjacent normal tissues in vivo. The knockdown of CASP4 significantly inhibited the invasion and migration but not the proliferation of PANC-1 cells. The knockdown of CHMP4C obviously inhibited the proliferation, migration, and invasion of PANC-1 cells. Conclusion: Pyroptosis-related genes play important roles in predicting the prognosis of PAAD, and CASP4 and CHMP4C affect the metastasis of PAAD.

CASP4 can be a diagnostic biomarker and correlated with immune infiltrates in gliomas.

Background: Gliomas are the most common and invasive malignant tumors that originate in the central nervous system. Currently, the primary treatment modality for gliomas is maximum surgical resection, supplemented by radiotherapy and chemotherapy. However, the long-term survival rate has not signifificantly increased. Pyroptosis is a new form of programmed lytic death that has been recently discovered. Caspase 4 (CASP4) plays a key role in pyroptosis. Many studies have shown that pyroptosis is not only related to inflflammation but is also closely related to the occurrence and development of most tumors. This study aimed to prove that CASP4 has a key role in the mechanism of gliomas. Methods: We used expression data from The Cancer Genome Atlas and the Chinese Glioma Genome Atlas to explore the relationship between CASP4 expression and glioma prognosis. The differential expression of CASP4 in gliomas and normal tissues was fifirst tested, and then the connection between CASP4 and tumor prognosis was explored. The relationship between CASP4 expression and immune cell infifiltration was also investigated. Finally, the possible pathways were analyzed using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis. Results: CASP4 was highly expressed and associated with a signifificantly lower survival rate in patients with glioma. It could also inflfluence immune cell infifiltration by releasing cytokines. Conclusion: CASP4 can be a diagnostic biomarker and is a promising therapeutic target for gliomas.

Prognostic autophagy model based on CASP4 and BIRC5 expression in patients with renal cancer: independent datasets-based study.

The purpose of this study was to identify key autophagy-related genes (ARGs) in patients with renal cancer (RC) by bioinformatics analysis, and to clarify their potential prognostic value. Thirty-eight differentially expressed ARGs were identified between RC and normal tissues based on The Cancer Genome Atlas database. Functional enrichment analysis suggested that autophagy may play a tumor-promoting role in the initiation of RC. We established a prognostic model with two ARGs (CASP4 and BIRC5) demonstrating significant correlations in expression levels with patient overall survival (OS). Multivariate Cox regression analysis showed that age and the autophagy genes prognostic model were independent prognostic factors for patients with RC. Considering the known prognostic significance of clinical stage in RC, we constructed a nomogram based on age, clinical stage, and the prognostic model. The prognostic model was verified in a separate validation set and external cohort of patients from Beijing Hospital. Patients of low and high risk were defined based on the median risk value calculated by the model and the high risk appeared associated with a significant shorter OS (P < 0.01). Overall, our findings reveal that ARGs have potential prognostic value in patients with RC, providing new directions for targeted therapy.

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection.

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.