Role Of Caspase-4 Research Articles

The Role of Caspase-4 and NLRP1 in MCF7 Cell Pyroptosis Induced by hUCMSC-Secreted Factors

Mesenchymal stem cells (MSCs) are being widely investigated for the development of novel therapeutic approaches for different cancers, including breast cancer, the leading form of cancer in women. Our previous study showed that the factors secreted by human umbilical cord MSCs (hUCMSCs) induced pyroptosis in the breast cancer cell line MCF7 and our RNA sequencing studies revealed an increase in the expression of the pyroptosis-related gene caspase-4 (CASP4) and nucleotide-binding, leucine-rich repeat pyrin domain-containing protein 1 (NLRP1) in pyroptotic MCF7 cells. Cellular pyroptosis can occur via the canonical pathway (involving caspase-1 and NLRP1) or the noncanonical pathway (involving caspase-4). In this study, we first confirmed that the inflammasome complex formed by NLRP1 and ASC is involved in MCF7 cell pyroptosis induced by hUCMSC-CM. Further, we investigated the role of CASP4 and NLRP1 in MCF7 cell pyroptosis induced by hUCMSC-secreted factors using shRNA-mediated transfection of CASP4 or NLRP1 in MCF7 cells. Cytotoxicity analyses revealed that neither CASP4 knockdown nor NLRP1 knockdown could inhibit the hUCMSC-CM-induced pyroptosis in MCF7 cells. Gene and protein expression analysis showed that hUCMSC-CM induced pyroptosis mainly via the canonical pathway in CASP4 knockdown MCF7 cells but mainly via the noncanonical pathway in NLRP1 knockdown MCF7 cells. Our study provides a foundation for further studies aimed at elucidating the precise mechanism underlying hUCMSC-induced pyroptosis in breast cancer cells and aid the identification of potential therapeutic targets for breast cancer.

CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion

Inflammatory caspases, including human caspase-4 (CASP4), play key roles in innate immune responses to promote fusion of phagosomes harboring pathogenic bacteria with lysosomes, halt intracellular replication of pathogens, maturation and secretion of pro-inflammatory cytokines. The role of inflammatory caspases in cancer cells remains poorly investigated. Here, we explored the consequences of modulating CASP4 expression levels on the migratory behavior of epithelial cancer cell lines. By a gene silencing approach and in vitro and in vivo studies we show that down-regulation of CASP4 leads to impaired cell migration and cell-matrix adhesion. This phenotype is accompanied by an increased actin cytoskeleton polymerization, changes in the overall organization of adherens junctions (AJs) and number and size of focal adhesions. Interestingly, the cell migration deficit could be reversed by epithelial growth factor treatment, and depletion of calcium ions unveiled a role of CASP4 in the novo assembly of AJs, suggesting that the role of CASP4 is not cell-autonomous. Finally, CASP4-silenced A431 cells exhibited a severe reduction in their ability to invade lung tissue, when injected into nude mice. Overall, our data support the emerging evidence that inflammatory caspases can regulate cell migration through actin remodeling and uncover a novel role of CASP4 in cancer cell behavior.

Entamoeba histolytica-induced IL-1β secretion is dependent on caspase-4 and gasdermin D.

During invasion, Entamoeba histolytica (Eh) encounter macrophages and activate them to elicit tissue damaging pro-inflammatory responses. When Eh binds macrophages via the Gal-lectin, surface EhCP-A5 RGD sequence ligates α5β1 integrin to activate caspase-1 in a complex known as the NLRP3 inflammasome. In this study, we investigated Eh requirements underlying macrophage caspase-4 and -1 activation and the role caspase-4 and gasdermin D (GSDMD) play in augmenting pro-inflammatory cytokine responses. Caspase-4 activation was similar to caspase-1 requiring live Eh attachment via the Gal-lectin and EhCP-A5. However, unlike caspase-1, caspase-4 activation was independent of ASC and NLRP3. Using CRISPR/Cas9 gene editing of caspase-4 and -1 and GSDMD, we determined that caspase-1 and bioactive IL-1β release was highly dependent on caspase-4 activation and cleavage of GSDMD in response to Eh. Formaldehyde cross-linking to stabilize protein-protein interactions in transfected COS-7 cells stimulated with Eh revealed that caspase-4 specifically interacted with caspase-1 in a protein complex that enhanced the cleavage of caspase-1 CARD domains to augment IL-1β release. Activated caspase-4 and -1 cleaved GSDMD liberating the N-terminal p30 pore-forming fragment that caused the secretion of IL-1β. These findings reveal a novel role for caspase-4 as a sensor molecule to amplify pro-inflammatory responses when macrophage encounters Eh.

Entamoeba histolytica‐Induced Caspase‐4/11 Activation Elicits a Pro‐Inflammatory Response in Macrophages

Entamoeba histolytica (Eh) is a protozoan parasite that colonizes in/on the mucus layer and in 10% of individuals, Eh invades the colonic mucosa to cause amebic colitis. A hallmark of amebiasis is acute intestinal inflammation dominated by the secretion of pro‐inflammatory cytokines, interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α. Eh in contact with macrophages activates caspase‐1 by the inflammasome complex resulting in the maturation of IL‐1β and ‐18. The role of inflammatory caspase‐4, ‐5, and ‐12 in humans are less understood. Caspase‐11, the murine homolog of caspase‐4, mediates non‐canonical inflammasome activation in response to particular pathogens, suggesting that caspase‐4 may have similar roles. As caspase‐4/11 might be an immune sensor in Eh infection the aim of this study was to identify a signature role for caspase‐4/11 in Eh‐induced macrophage responses. To determine if caspase‐4/11 was involved in inflammasome signalling, the gene was silenced with siRNA. Knockdown of caspase‐4/11 did not affect caspase‐1 activation or IL‐1β secretion in response to Eh. However, multiplex cytokine arrays showed marked up regulation of TNF‐α, MIP‐1β, and KC suggesting that caspase‐4/11 may have regulatory roles in the secretion of distinct cytokines/chemokines. Although there was no evidence for crosstalk between caspase‐4/11 and caspase‐1, these inflammatory caspases seem to play a critical role in regulating other cytokines/chemokines that shape the overall macrophage response towards Eh infection. Elucidating the role these inflammatory caspases play in Eh disease pathogenesis may shed light on new therapeutics to treat amebiasis.Grant Support: NSERC

Abstract 2717: Reolysin: A novel reovirus-based agent that induces endoplasmic reticular stress in RAS-activated pancreatic cancer

Abstract Mutations in the RAS oncogene are associated with transformation and proliferation and are prevalent in many tumor types. Since RAS is frequently hyperactivated in pancreatic cancer and plays an essential role in tumor progression, it is an ideal therapeutic target. Reoviruses are naturally occurring viruses that are non-pathogenic and have been reported to specifically replicate in cancer cells with an activated RAS pathway, but not in normal tissue. To take advantage of this observation therapeutically, the reovirus-based anticancer agent Reolysin was recently developed. Reolysin has already progressed into clinical trials, but its mechanisms of action have not been fully elucidated. We hypothesized that Reolysin exposure would result in the production of viral products, stimulate endoplasmic reticular (ER) stress, and induce apoptosis. Reolysin treatment triggered the selective replication of reoviruses as visualized by immunocytochemistry and electron microscopy in KRAS-transformed immortalized human pancreatic duct epithelial cells and in a panel of pancreatic cancer cell lines. Reovirus accumulation was associated with an increase in the expression of ER stress-related genes as assessed by microarray and quantitative RT-PCR analyses, cleavage of caspase-4, and induction of apoptosis. In support of our hypothesis that Reolysin induces ER stress-mediated apoptosis, co-treatment with tunicamycin or brefeldin A further augmented Reolysin-induced cell death. Previous studies have demonstrated that the proteasome inhibitor bortezomib causes ER stress-mediated apoptosis. Here we show that co-treatment with bortezomib and Reolysin stimulates the simultaneous accumulation of ubiquitinated and viral proteins resulting in enhanced levels of ER stress-related gene expression, caspase-4 cleavage, and apoptosis. The role of caspase-4 as a regulator of therapeutic sensitivity to these agents was investigated by targeting caspase-4 expression with siRNA. Knockdown of caspase-4 blunted apoptosis stimulated by Reolysin, bortezomib, and the combination. Importantly, bortezomib also dramatically augmented the anticancer activity of Reolysin in xenograft models of pancreatic cancer in vivo. Analysis of tumor specimens from mice treated with Reolysin and bortezomib displayed reovirus accumulation, enhanced expression of GRP78/Bip, a marker of ER stress and increased levels of apoptosis. Collectively, our results demonstrate that the abnormal protein accumulation induced by Reolysin and bortezomib promotes heightened ER stress and apoptosis in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2717. doi:1538-7445.AM2012-2717

Caspase-4 may play a role in loss of proximal tubules and renal injury in nephropathic cystinosis

Nephropathic cystinosis is characterized clinically by generalized proximal renal tubular dysfunction, renal Fanconi Syndrome and progressive renal failure. Glomerular-proximal tubule disconnection has been noted in renal biopsies from patients with nephropathic cystinosis. In vitro studies performed in cystinotic fibroblasts and renal proximal tubular cells support a role for apoptosis of the glomerulotubular junction, and we have further extended these studies to human native cystinotic kidney specimens. We performed semi-quantitative analysis of tubular density in kidney biopsies from patients with nephropathic cystinosis and demonstrated a significant reduction (p=0.0003) in the number of proximal tubules in the kidney tissue of patients with cystinosis compared to normal kidneys and kidneys with other causes of renal injury; this reduction appears to be associated with the over-expression of caspase-4. This study provides the first quantitative evidence of a loss of proximal tubules in nephropathic cystinosis and suggests a possible role of caspase-4 in the apoptotic loss of proximal tubular cells. Further work is needed to elucidate if this injury mechanism may be causative for the progression of renal functional decline in nephropathic cystinosis.