The stressosome, a caspase-8-activating signalling complex assembled in response to cell stress in an ATG5-mediated manner.

Stress-induced apoptosis is mediated primarily through the intrinsic pathway that involves caspase-9. We previously reported that in caspase-9-deficient cells, a protein complex containing ATG5 and Fas-associated death domain (FADD) facilitated caspase-8 activation and cell death in response to endoplasmic reticulum (ER) stress. Here, we investigated whether this complex could be activated by other forms of cell stress. We show that diverse stress stimuli, including etoposide, brefeldin A and paclitaxel, as well as heat stress and gamma-irradiation, caused formation of a complex containing ATG5-ATG12, FADD and caspase-8 leading to activation of downstream caspases in caspase-9-deficient cells. We termed this complex the 'stressosome'. However, in these cells, only ER stress and heat shock led to stressosome-dependent cell death. Using in silico molecular modelling, we propose the structure of the stressosome complex, with FADD acting as an adaptor protein, interacting with pro-caspase-8 through their respective death effector domains (DEDs) and interacting with ATG5-ATG12 through its death domain (DD). This suggests that the complex could be regulated by cellular FADD-like interleukin-1ß-converting enzyme-inhibitory protein (cFLIPL ), which was confirmed experimentally. This study provides strong evidence for an alternative mechanism of caspase-8 activation involving the stressosome complex.

Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells.

Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as "find-me" signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.

The role of the unfolded protein response in cancer progression: From oncogenesis to chemoresistance.

Tumour cells endure both oncogenic and environmental stresses during cancer progression. Transformed cells must meet increased demands for protein and lipid production needed for rapid proliferation and must adapt to exist in an oxygen- and nutrient-deprived environment. To overcome such challenges, cancer cells exploit intrinsic adaptive mechanisms such as the unfolded protein response (UPR). The UPR is a pro-survival mechanism triggered by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1, PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress, they signal in a coordinated fashion to re-establish ER homoeostasis, thus aiding cell survival. Over the past decade, evidence has emerged supporting a role for the UPR in the establishment and progression of several cancers, including breast cancer, prostate cancer and glioblastoma multiforme. This review discusses our current knowledge of the UPR during oncogenesis, tumour growth, metastasis and chemoresistance.

Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α.

Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.

RIP2 enhances cell survival by activation of NF-ĸB in triple negative breast cancer cells.

Receptor-interacting protein 2 (RIP2) is an essential mediator of inflammation and innate immunity, but little is known about its role outside the immune system. Recently, RIP2 has been linked to chemoresistance of triple negative breast cancer (TNBC), the most aggressive breast cancer subtype for which there is an urgent need for targeted therapies. In this study we show that high expression of RIP2 in breast tumors correlates with a worse prognosis and a higher risk of recurrence. We also demonstrate that RIP2 confers TNBC cell resistance against paclitaxel and ceramide-induced apoptosis. Overexpression of RIP2 lead to NF-κB activation, which contributed to higher expression of pro-survival proteins and cell survival. Conversely, RIP2 knockdown inhibited NF-κB signaling, reduced levels of anti-apoptotic proteins and sensitized cells to drug treatment. Together, these data show that RIP2 promotes survival of breast cancer cells through NF-κB activation and that targeting RIP2 may be therapeutically beneficial for treatment of TNBC.

BCL-2 modulates the unfolded protein response by enhancing splicing of X-box binding protein-1.

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) triggers a highly conserved stress response mechanism termed the unfolded protein response (UPR). The UPR is a complex series of signaling pathways controlled by ER localized transmembrane receptors, PERK, ATF6 and IRE1α. Following activation IRE1α splices XBP-1 mRNA facilitating the formation of a potent transcription factor, spliced XBP-1. The BCL-2 family members, BAX and BAK, in addition to the mitochondrion also localize to the ER and have been demonstrated to directly interact with IRE1α promoting its activity. In this study we show that in addition to BAX and BAK, the anti-apoptotic BCL-2 protein can regulate IRE1α activity. Enhanced splicing of XBP-1 was observed in BCL-2 overexpressing cells implicating BCL-2 in the complex regulation of IRE1α activity.

Inhibitor of apoptosis proteins (IAPs) and their antagonists regulate spontaneous and tumor necrosis factor (TNF)-induced proinflammatory cytokine and chemokine production.

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.

Stressed to death - mechanisms of ER stress-induced cell death.

The endoplasmic reticulum (ER) is a highly dynamic organelle of fundamental importance present in all eukaryotic cells. The majority of synthesized structural and secreted proteins undergo post-translational modification, folding and oligomerization in the ER lumen, enabling proteins to carry out their physiological functions. Therefore, maintenance of ER homeostasis and function is imperative for proper cellular function. Physiological and pathological conditions can disturb ER homeostasis and thus negatively impact upon protein folding, resulting in an accumulation of unfolded proteins. Examples include hypoxia, hypo- and hyperglycemia, acidosis, and fluxes in calcium levels. Increased levels of unfolded/misfolded proteins within the ER lumen triggers a condition commonly referred to as 'ER stress'. To combat ER stress, cells have evolved a highly conserved adaptive stress response referred to as the unfolded protein response (UPR). UPR signaling affords the cell a 'window of opportunity' for stress resolution however, if prolonged or excessive the UPR is insufficient and ER stress-induced cell death ensues. This review discusses the role of ER stress sensors IRE1, PERK and ATF6, describing their role in ER stress-induced death signaling with specific emphasis placed upon the importance of the intrinsic cell death pathway and Bcl-2 family regulation.

IRE1 signaling increases PERK expression during chronic ER stress.

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses.

New directions in ER stress-induced cell death.

Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of many diseases including heart disease, cancer and neurodegenerative diseases such as Alzheimer's and Huntington's. Prolonged or excessive ER stress results in the initiation of signaling pathways resulting in cell death. Over the past decade much research investigating the onset and progression of ER stress-induced cell death has been carried out. Owing to this we now have a better understanding of the signaling pathways leading to ER stress-mediated cell death and have begun to appreciate the importance of ER localized stress sensors, IRE1α, ATF6 and PERK in this process. In this article we provide an overview of the current thinking and concepts concerning the various stages of ER stress-induced cell death, focusing on the role of ER localized proteins in sensing and triggering ER stress-induced death signals with particular emphasis on the contribution of calcium signaling and Bcl-2 family members to the execution phase of this process. We also highlight new and emerging directions in ER stress-induced cell death research particularly the role of microRNAs, ER-mitochondria cross talk and the prospect of mitochondria-independent death signals in ER stress-induced cell death.

Unfolding the Interactions between Endoplasmic Reticulum Stress and Oxidative Stress.

Oxidative stress is caused by an imbalance in cellular redox state due to the accumulation of reactive oxygen species (ROS). While homeostatic levels of ROS are important for cell physiology and signaling, excess ROS can induce a variety of negative effects ranging from damage to biological macromolecules to cell death. Additionally, oxidative stress can disrupt the function of redox-sensitive organelles including the mitochondria and endoplasmic reticulum (ER). In the case of the ER, the accumulation of misfolded proteins can arise due to oxidative stress, leading to the onset of ER stress. To combat ER stress, cells initiate a highly conserved stress response called the unfolded protein response (UPR). While UPR signaling, within the context of resolving ER stress, is well characterised, how UPR mediators respond to and influence oxidative stress is less defined. In this review, we evaluate the interplay between oxidative stress, ER stress and UPR signaling networks. Specifically, we assess how UPR signaling mediators can influence antioxidant responses.

DUBing Primary Tumors of the Central Nervous System: Regulatory Roles of Deubiquitinases.

The ubiquitin proteasome system (UPS) utilizes an orchestrated enzymatic cascade of E1, E2, and E3 ligases to add single or multiple ubiquitin-like molecules as post-translational modification (PTM) to proteins. Ubiquitination can alter protein functions and/or mark ubiquitinated proteins for proteasomal degradation but deubiquitinases (DUBs) can reverse protein ubiquitination. While the importance of DUBs as regulatory factors in the UPS is undisputed, many questions remain on DUB selectivity for protein targeting, their mechanism of action, and the impact of DUBs on the regulation of diverse biological processes. Furthermore, little is known about the expression and role of DUBs in tumors of the human central nervous system (CNS). In this comprehensive review, we have used publicly available transcriptional datasets to determine the gene expression profiles of 99 deubiquitinases (DUBs) from five major DUB families in seven primary pediatric and adult CNS tumor entities. Our analysis identified selected DUBs as potential new functional players and biomarkers with prognostic value in specific subtypes of primary CNS tumors. Collectively, our analysis highlights an emerging role for DUBs in regulating CNS tumor cell biology and offers a rationale for future therapeutic targeting of DUBs in CNS tumors.

Caspase-1 promiscuity is counterbalanced by rapid inactivation of processed enzyme.

Members of the caspase family of cysteine proteases coordinate the highly disparate processes of apoptosis and inflammation. However, although hundreds of substrates for the apoptosis effector caspases (caspase-3 and caspase-7) have been identified, only two confirmed substrates for the key inflammatory protease (caspase-1) are known. Whether this reflects intrinsic differences in the substrate specificity of inflammatory versus apoptotic caspases or their relative abundance in vivo is unknown. To address this issue, we have compared the specificity of caspases-1, -3, and -7 toward peptide and protein substrates. Contrary to expectation, caspase-1 displayed concentration-dependent promiscuity toward a variety of substrates, suggesting that caspase-1 specificity is maintained by restricting its abundance. Although endogenous concentrations of caspase-1 were found to be similar to caspase-3, processed caspase-1 was found to be much more labile, with a half-life of ~9 min. This contrasted sharply with the active forms of caspase-3 and caspase-7, which exhibited half-lives of 8 and 11 h, respectively. We propose that the high degree of substrate specificity displayed by caspase-1 is maintained through rapid spontaneous inactivation of this protease.

New insights into IRE1α activation and function in anti-tumor immunity.

Logue, Gorman, and Samali highlight a study by Guttman and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202111068) that shows exogenous antigen peptides imported into the ER can activate the ER stress sensor IRE1α, attenuating cross-presentation by dendritic cells.

An Emerging Role for the Unfolded Protein Response in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and one of the leading causes of cancer-associated deaths in the world. It is characterised by dismal response rates to conventional therapies. A major challenge in treatment strategies for PDAC is the presence of a dense stroma that surrounds the tumour cells, shielding them from treatment. This unique tumour microenvironment is fuelled by paracrine signalling between pancreatic cancer cells and supporting stromal cell types including the pancreatic stellate cells (PSC). While our molecular understanding of PDAC is improving, there remains a vital need to develop effective, targeted treatments. The unfolded protein response (UPR) is an elaborate signalling network that governs the cellular response to perturbed protein homeostasis in the endoplasmic reticulum (ER) lumen. There is growing evidence that the UPR is constitutively active in PDAC and may contribute to the disease progression and the acquisition of resistance to therapy. Given the importance of the tumour microenvironment and cytokine signalling in PDAC, and an emerging role for the UPR in shaping the tumour microenvironment and in the regulation of cytokines in other cancer types, this review explores the importance of the UPR in PDAC biology and its potential as a therapeutic target in this disease.

Multiplex Serum Biomarker Assays Improve Prediction of Renal and Mortality Outcomes in Chronic Kidney Disease.

BACKGROUND: We investigated the predictive value of 11 serum biomarkers for renal and mortality end points in people with CKD. METHODS: Adults with CKD (n=139) were enrolled from outpatient clinics between February 2014 and November 2016. Biomarker quantification was performed using two multiplex arrays on a clinical-grade analyzer. Relationships between biomarkers and renal and mortality end points were investigated by random forests and Cox proportional hazards regression. RESULTS: The cohort was 56% male. The mean age was 63 years and median (IQR) CKD-EPI eGFR was 33 (24-51) ml/min per BSA. A total of 56 (40%) people developed a composite end point defined as ≥40% decline in eGFR, doubling of serum creatinine, RRT, or death over median (IQR) follow-up of 5.4 (4.7-5.7) years. Prediction of the composite end point was better with random forests trained on serum biomarkers compared with clinical variables (area under the curve of 0.81 versus 0.78). The predictive performance of biomarkers was further enhanced when considered alongside clinical variables (area under the curve of 0.83 versus 0.81 for biomarkers alone). Patients (n=27, 19%) with high soluble TNF receptor-1 (≥3 ng/ml) and neutrophil gelatinase-associated lipocalin (≥156 ng/ml), coupled with low complement 3a des-arginine (<2368 ng/ml), almost universally (96%) developed the composite renal and mortality end point. C-reactive protein (adjusted hazard ratio, 1.4; 95% CI, 1.1 to 1.8), neutrophil gelatinase-associated lipocalin (adjusted hazard ratio, 2.8; 95% CI, 1.3 to 6.1) and complement 3a desarginine (adjusted hazard ratio, 0.6; 95% CI, 0.4 to 0.96) independently predicted time to the composite end point. CONCLUSIONS: Outpatients with the triad of high soluble TNF receptor-1 and neutrophil gelatinase-associated lipocalin coupled with low complement 3a des-arginine had high adverse event rates over 5-year follow-up. Incorporation of serum biomarkers alongside clinical variables improved prediction of CKD progression and mortality. Our findings require confirmation in larger, more diverse patient cohorts.

Tumour Cell Secretome in Chemoresistance and Tumour Recurrence.

Chemoresistance is a major factor driving tumour relapse and the high rates of cancer-related deaths. Understanding how cancer cells overcome chemotherapy-induced cell death is critical in promoting patient survival. One emerging mechanism of chemoresistance is the tumour cell secretome (TCS), an array of protumorigenic factors released by tumour cells. Chemotherapy exposure can also alter the composition of the TCS, known as therapy-induced TCS, and can promote tumour relapse and the formation of an immunosuppressive tumour microenvironment (TME). Here, we outline how the TCS can protect cancer cells from chemotherapy-induced cell death. We also highlight recent evidence describing how therapy-induced TCS can impact cancer stem cell (CSC) expansion and tumour-associated immune cells to enable tumour regrowth and antitumour immunity.

Correction: Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

The inflammasome is a multiprotein complex assembled in response to Pathogen Associated Molecular Patterns (PAMPs) and Danger Associated Molecular Patterns (DAMPs). Inflammasome activation occurs through a two-step mechanism, with the first signal facilitating priming of inflammasome components while the second signal triggers complex assembly. Once assembled, the inflammasome recruits and activates pro-caspase-1, which in turn processes pro-interleukin (IL)-18 and pro-IL-1ß into their bio-active forms. Owing to its key role in the regulation of innate immune responses, the inflammasome has emerged as a therapeutic target for the treatment of inflammatory conditions. In this study we demonstrate that IRE1α, a key component of the Unfolded Protein Response, contributes to assembly of the NLRP3 inflammasome. Blockade of IRE1α RNase signaling lowered NLRP3 inflammasome assembly, caspase-1 activation and pro-IL-1ß processing. These results underscore both the importance and potential therapeutic relevance of targeting IRE1α signaling in conditions of excessive inflammasome formation.

The Unfolded Protein Response in Breast Cancer.

In 2018, in the US alone, it is estimated that 268,670 people will be diagnosed with breast cancer, and that 41,400 will die from it. Since breast cancers often become resistant to therapies, and certain breast cancers lack therapeutic targets, new approaches are urgently required. A cell-stress response pathway, the unfolded protein response (UPR), has emerged as a promising target for the development of novel breast cancer treatments. This pathway is activated in response to a disturbance in endoplasmic reticulum (ER) homeostasis but has diverse physiological and disease-specific functions. In breast cancer, UPR signalling promotes a malignant phenotype and can confer tumours with resistance to widely used therapies. Here, we review several roles for UPR signalling in breast cancer, highlighting UPR-mediated therapy resistance and the potential for targeting the UPR alone or in combination with existing therapies.