Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate.

The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.

Aggressive migration in acidic pH of a glioblastoma cancer stem cell line in vitro is independent of ASIC and KCa3.1 ion channels, but involves phosphoinositide 3-kinase.

The microenvironment of proliferative and aggressive tumours, such as the brain tumour glioblastoma multiforme (GBM), is often acidic, hypoxic, and nutrient deficient. Acid-sensing ion channels (ASICs) are proton-sensitive Na+ channels that have been proposed to play a role in pH sensing and in modulation of cancer cell migration. We previously reported that primary glioblastoma stem cells (GSCs), which grow as multicellular tumour spheroids, express functional ASIC1a and ASIC3, whereas ASIC2a is downregulated in GSCs. Using a 2.5D migration assay, here we report that acidic pH dramatically increased migration of GSCs of the pro-neural subtype. Pharmacological blockade as well as CRISPR-Cas9-mediated gene knock-out of ASIC1a or stable overexpression of ASIC2a, however, revealed that neither ASIC1a nor ASIC3, nor downregulation of ASIC2a, mediated the aggressive migration at acidic pH. Therefore, we tested the role of two other proteins previously implicated in cancer cell migration: the Ca2+-activated K+ channel KCa3.1 (KCNN4) and phosphoinositide 3-kinase (PI3K). While pharmacological blockade of KCa3.1 did also not affect migration, blockade of PI3K decreased migration at acidic pH to control levels. In summary, our study reveals a strongly enhanced migration of GSCs at acidic pH in vitro and identifies PI3K as an important mediator of this effect.

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.

RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation.

TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.

TNF Is Partially Required for Cell-Death-Triggered Skin Inflammation upon Acute Loss of cFLIP.

cFLIP is required for epidermal integrity and skin inflammation silencing via protection from TNF-induced keratinocyte apoptosis. Here, we generated and analyzed cFLIP epidermal KO mice with additional TNF deficiency. Intriguingly, the ablation of TNF rescued the pathological phenotype of epidermal cFLIP KO from characteristic weight loss and increased mortality. Moreover, the lack of TNF in these animals strongly reduced and delayed the epidermal hyperkeratosis and the increased apoptosis in keratinocytes. Our data demonstrate that TNF signaling in cFLIP-deficient keratinocytes is the critical factor for the regulation of skin inflammation via modulated cytokine and chemokine expression and, thus, the attraction of immune cells. Our data suggest that autocrine TNF loop activation upon cFLIP deletion is dispensable for T cells, but is critical for neutrophil attraction. Our findings provide evidence for a negative regulatory role of cFLIP for TNF-dependent apoptosis and partially for epidermal inflammation. However, alternative signaling pathways may contribute to the development of the dramatic skin disease upon cFLIP deletion. Our data warrant future studies of the regulatory mechanism controlling the development of skin disease upon cFLIP deficiency and the role of cFLIP/TNF in a number of inflammatory skin diseases, including toxic epidermal necrolysis (TEN).

cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms.

The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either apoptosis or necroptosis. This 2 MDa intracellular complex that we designate "Ripoptosome" is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIP(L) prevents Ripoptosome formation, whereas, intriguingly, cFLIP(S) promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the "rheostat" that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.

Overcoming cell death resistance in skin cancer therapy: Novel translational perspectives.

In the last decade, significant progress has been made in understanding skin cancer cell death resistance mechanisms, and a number of new treatment strategies have been developed. Systematic approach genomic studies of various cancer types have opened new possibilities for the development of anticancer therapies. However, there are still fundamental gaps in the challenging biomedical puzzle, which will form a complete picture for curing cancer. Thus, herein, we describe some of the current cancer treatment strategies and discuss additional cell signalling pathways that could be potential targets for skin cancer treatment.

Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex.

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions.

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn't rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.

Pitfalls in the Application of Dispase-Based Keratinocyte Dissociation Assay for In Vitro Analysis of Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is a chronic, life-altering autoimmune disease due to the production of anti-desmoglein antibodies causing the loss of cell-cell adhesion in keratinocytes (acantholysis) and blister formation in both skin and mucous membranes. The dispase-based keratinocyte dissociation assay (DDA) is the method of choice to examine the pathogenic effect of antibodies and additional co-stimuli on cell adhesion in vitro. Despite its widespread use, there is a high variability of experimental conditions, leading to inconsistent results. In this paper, we identify and discuss pitfalls in the application of DDA, including generation of a monolayer with optimized density, appropriate culturing conditions to obtain said monolayer, application of mechanical stress in a standardized manner, and performing consistent data processing. Importantly, we describe a detailed protocol for a successful and reliable DDA and the respective ideal conditions for three different types of human keratinocytes: (1) primary keratinocytes, (2) the HaCaT spontaneously immortalized keratinocyte cell line, and (3) the recently characterized HaSKpw spontaneously immortalized keratinocyte cell line. Our study provides detailed protocols which guarantee intra- and inter-experimental comparability of DDA.

Helicobacter pylori-induced activation of beta-catenin involves low density lipoprotein receptor-related protein 6 and Dishevelled.

BACKGROUND: The human microbial pathogen Helicobacter pylori resides in the stomach of about fifty percent of the world's population and represents a risk factor for chronic gastritis, peptic ulcers and, in rare cases, gastric cancer. Alterations of the Wnt/beta-catenin signaling pathway have been described in almost every human cancer disease, due to the regulation of target genes being involved in cell cycle control, differentiation, cell migration or stem cell control. Our study aimed to elucidate the role of proximal Wnt signaling components low density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled (Dvl) in the activation of beta-catenin early after infection of gastric epithelial cells with H. pylori. RESULTS: Infection of gastric epithelial NCI-N87 cells with H. pylori induces rapid phosphorylation of the Wnt/beta-catenin pathway co-receptor LRP6 independent of the cytotoxin-associated gene A (CagA) or vacuolating cytotoxin A (VacA). However, bacteria lacking a functional type 4 secretion system (T4SS) failed to induce LRP6 phosphorylation. Further, we identified proteins of the Dvl family, namely Dvl2 and Dvl3, which are involved in LRP6 phosphorylation. H. pylori-induced nuclear accumulation of beta-catenin and its transcriptional activation, and expression of Wnt target genes are strongly reduced in stable knockdown cell lines deficient for LRP6, Dvl2 or Dvl3. CONCLUSION: We analysed the H. pylori-induced activation of Wnt-signaling factors and demonstrate for the first time that the canonical Wnt-signaling proteins LRP6 and Dvl2 and Dvl3 are involved in the regulation of beta-catenin.

A20 Promotes Ripoptosome Formation and TNF-Induced Apoptosis via cIAPs Regulation and NIK Stabilization in Keratinocytes.

The ubiquitin-editing protein A20 (TNFAIP3) is a known key player in the regulation of immune responses in many organs. Genome-wide associated studies (GWASs) have linked A20 with a number of inflammatory and autoimmune disorders, including psoriasis. Here, we identified a previously unrecognized role of A20 as a pro-apoptotic factor in TNF-induced cell death in keratinocytes. This function of A20 is mediated via the NF-κB-dependent alteration of cIAP1/2 expression. The changes in cIAP1/2 protein levels promote NIK stabilization and subsequent activation of noncanonical NF-κB signaling. Upregulation of TRAF1 expression triggered by the noncanonical NF-κB signaling further enhances the NIK stabilization in an autocrine manner. Finally, stabilized NIK promotes the formation of the ripoptosome and the execution of cell death. Thus, our data demonstrate that A20 controls the execution of TNF-induced cell death on multiple levels in keratinocytes. This signaling mechanism might have important implications for the development of new therapeutic strategies for the treatment of A20-associated skin diseases.

TAK1 suppresses RIPK1-dependent cell death and is associated with disease progression in melanoma.

Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies as well as immunotherapies frequently remain inefficient. Alternative means to treat melanoma, in particular through the induction of programmed cell death modalities such as apoptosis or necroptosis, therefore still need to be explored. Here, we report that melanoma cell lines expressing notable amounts of RIPK1, RIPK3 and MLKL, the key players of necroptosis signal transduction, fail to execute necroptotic cell death. Interestingly, the activity of transforming growth factor ß-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5Z)-7-Oxozeaenol, deletion of MAP3K7 or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNFα or TRAIL based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5Z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Correspondingly, elevated expression of TAK1 correlates with reduced disease free survival in patients diagnosed with primary melanoma. Overall, our results therefore demonstrate that TAK1 suppresses the susceptibility to RIPK1-dependent cell death and that high expression of TAK1 indicates an increased risk for disease progression in melanoma.

RIPK1 Suppresses a TRAF2-Dependent Pathway to Liver Cancer.

Receptor-interacting protein kinase 1 (RIPK1) represents an essential signaling node in cell death and inflammation. Ablation of Ripk1 in liver parenchymal cells (LPC) did not cause a spontaneous phenotype, but led to tumor necrosis factor (TNF)-dependent hepatocyte apoptosis and liver injury without affecting inducible nuclear factor κB (NF-κB) activation. Loss of Ripk1 induced the TNF-dependent proteasomal degradation of the E3-ligase, TNF receptor-associated factor 2 (TRAF2), in a kinase-independent manner, thereby activating caspase-8. Moreover, loss of both Ripk1 and Traf2 in LPC not only resulted in caspase-8 hyperactivation but also impaired NF-κB activation, promoting the spontaneous development of hepatocellular carcinoma. In line, low RIPK1 and TRAF2 expression in human HCCs was associated with an unfavorable prognosis, suggesting that RIPK1 collaborates with TRAF2 to inhibit murine and human hepatocarcinogenesis.

Ripoptosome Analysis by Caspase-8 Coimmunoprecipitation.

The biochemical signaling of cell death pathways is executed at a number of different intracellular and/or membrane-bound high-molecular mass complexes. It is crucial to be able to detect the formation, differences in assembly, and differential composition of such complexes to understand their contribution to the execution phase of apoptotic or necroptotic cell death. We describe here the use of caspase-8 coimmunoprecipitation in the spontaneously transformed keratinocyte cell line, HaCaT, to study the formation and composition of the Ripoptosome, a complex that is based on the serine-threonine kinase receptor-interacting protein 1 (RIPK1). However, the method can be adapted for use with other antibodies and cell lines. This protocol determines whether cells form the Ripoptosome complex, which is important for both apoptosis and necroptosis execution. Caspase-8 is an indispensible Ripoptosome component; therefore, caspase-8 antibodies are used to pull down the respective complex. However, the method cannot discriminate whether this complex triggers apoptosis (through the RIPK1 → FADD → caspase-8 activation pathway), necroptosis (through the RIPK1 → RIPK3 → MLKL activation pathway) or nondeath signaling. The actual signaling output (death or nondeath signaling) depends on the stoichiometry of the respective molecules as well as on the activity of FLIP, caspase-8, or other factors.

Crystal Violet Assay for Determining Viability of Cultured Cells.

Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere.

Techniques to Distinguish Apoptosis from Necroptosis.

The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis.

Programmed necrosis and necroptosis signalling.

In recent years, the paradigm of cell death regulation has changed. Nowadays, not only apoptosis but also several forms of necrosis (e.g. necroptosis) are considered to be regulated. The central roles of receptor-interacting serine/threonine protein kinase1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein, and the molecular signalling platforms in which these molecules participate, are being intensively studied. In particular, the role of RIPK1, being both a kinase and a scaffold molecule, in different cell death regulatory complexes is of great relevance for the field. This minireview aims to introduce the emerging and dynamic field of necroptosis to the reader, with a specific focus on intracellular signalling pathways involved in this process.

Silencing autocrine death: a ubiquitin ligase that blocks activation-induced cell death in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) tumor cells lack the ability of activated T cells to undergo TCR/CD3-mediated activation-induced cell death (AICD). In this issue, the study reported by Wu et al. demonstrates that c-CBL (Casitas B-lineage Lymphoma proto-oncogene) is overexpressed in CTCL. When CTCL cells lose c-CBL, AICD is enhanced. Furthermore, combination therapy with methotrexate (a known demethylating agent for the CD95 gene) in combination with the loss of c-CBL increases CTCL cell death. Therefore, inhibition of c-CBL could represent a method of sensitizing lymphoma cells to enhance AICD. Armed with their novel data, the investigators envision combination therapies that target c-CBL to reactivate AICD in the malignant T cells whenever responsiveness to TCR/CD3 signaling is retained.