Death Effector Research Articles

The molluscum contagiosum virus death effector domain containing protein MC160 RxDL motifs are not required for its known viral immune evasion functions

The molluscum contagiosum virus (MCV) uses a variety of immune evasion strategies to antagonize host immune responses. Two MCV proteins, MC159 and MC160, contain tandem death effector domains (DEDs). They are reported to inhibit innate immune signaling events such as NF-κB and IRF3 activation, and apoptosis. The RxDL motif of MC159 is required for inhibition of both apoptosis and NF-κB activation. However, the role of the conserved RxDL motif in the MC160 DEDs remained unknown. To answer this question, we performed alanine mutations to neutralize the arginine and aspartate residues present in the MC160 RxDL in both DED1 and DED2. These mutations were further modeled against the structure of the MC159 protein. Surprisingly, the RxDL motif was not required for MC160's ability to inhibit MAVS-induced IFNβ activation. Further, unlike previous results with the MC159 protein, mutations within the RxDL motif of MC160 had no effect on the ability of MC160 to dampen TNF-α-induced NF-κB activation. Molecular modeling predictions revealed no overall changes to the structure in the MC160 protein when the amino acids of both RxDL motifs were mutated to alanine (DED1=R67A D69A; DED2=R160A D162A). Taken together, our results demonstrate that the RxDL motifs present in the MC160 DEDs are not required for known functions of the viral protein.

Abstract 59: Post-stroke Delivery of Microrna-363 to Middle-aged Female Rats is Internalized by Neurons and Suppresses the Cell Death Effector Pathway

Background: MicroRNAs serve as translational inhibitors and offer a unique therapeutic target for acute diseases such as stroke. Profiling of circulating miRNA after stroke identified mir363, whose expression was inversely correlated with infarct volume. Middle aged female rats show worse stroke outcomes than younger females and have much lower levels of mir363. Our recent studies showed that iv injections of miR363 mimic to middle aged females, significantly improved stroke outcome. The present study is designed to determine the mechanisms by which miR-363 acts as a therapeutic miR. Methods: Middle aged (12 mo) females were subject to MCAo. At 4h post-stroke, animals received a tail-vein injection of miR-363-3p FAM or scrambled control. Animals were terminated at 48h or 5d post-MCAo and perfused transcardially or processed for protein, respectively. To determine which neural cell types localized exogenous mir363-3p, combined immunofluorescence was performed for cell specific markers (neuronal (NeuN), astrocytic (GFAP), microglial (CD11b) and endothelial (PECAM)) and mir363-3p-FAM mimic on coronal brain sections (25 mm thickness). Protein lysates from the ischemic tissue was analyzed for caspase-3 expression by Western blot analysis. Results: FAM-labeled mir363-3p was widely detected in the forebrain. The majority of NeuN+ cells in the cortex and striatum were also labeled with FAM-363-3p, indicating a robust internalization of the mimic in neurons. FAM-mir363-3p was also localized to a few microglia (CD11b +), virtually no double-label was seen in astrocytes and endothelial cells. Mir363 decreased the expression and functional activity of caspase3 in the ischemic hemisphere. Conclusion: Collectively, the data suggests that exogenous miR-363-3p is shuttled to the brain and is preferentially internalized by neurons. Together with the caspase-3 regulation, our data suggests that mir363 may improve stroke outcomes by suppressing a cell death effector.

Apolipoprotein CIII Overexpression-Induced Hypertriglyceridemia Increases Nonalcoholic Fatty Liver Disease in Association with Inflammation and Cell Death.

Nonalcoholic fatty liver disease (NAFLD) is the principal manifestation of liver disease in obesity and metabolic syndrome. By comparing hypertriglyceridemic transgenic mice expressing apolipoprotein (apo) CIII with control nontransgenic (NTg) littermates, we demonstrated that overexpression of apoCIII, independent of a high-fat diet (HFD), produces NAFLD-like features, including increased liver lipid content; decreased antioxidant power; increased expression of TNFα, TNFα receptor, cleaved caspase-1, and interleukin-1β; decreased expression of adiponectin receptor-2; and increased cell death. This phenotype is aggravated and additional NAFLD features are differentially induced in apoCIII mice fed a HFD. HFD induced glucose intolerance together with increased gluconeogenesis, indicating hepatic insulin resistance. Additionally, the HFD led to marked increases in plasma TNFα (8-fold) and IL-6 (60%) in apoCIII mice. Cell death signaling (Bax/Bcl2), effector (caspase-3), and apoptosis were augmented in apoCIII mice regardless of whether a HFD or a low-fat diet was provided. Fenofibrate treatment reversed several of the effects associated with diet and apoCIII expression but did not normalize inflammatory traits even when liver lipid content was fully corrected. These results indicate that apoCIII and/or hypertriglyceridemia plays a major role in liver inflammation and cell death, which in turn increases susceptibility to and the severity of diet-induced NAFLD.

Analysis of Argonaute Complex Bound mRNAs in DU145 Prostate Carcinoma Cells Reveals New miRNA Target Genes.

Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs.

Cflarb Complemented the Function of Cflara to Allow Cflara Knock out Zebrafish To Normal Development

Cellular FLICE-inhibitory protein (cFLIP, cflara) is a regulator of death receptor (DR)-induced apoptosis and NF-κB activation. cFLIP is known to prevent activation of the caspase cascade by binding to FADD/caspase-8. Up-regulated cFLIP has been identified in many tumor types, and therefore restoring apoptosis by silencing cFLIP may be one of the more potent strategies in cancer therapeutics. The zebrafish cFLIP gene, cflara, has 2 death effector domains (DEDs) and a single caspase-like domain. Expression of cflara was detected in the zebrafish embryo by RT-PCR and whole-mount in situ hybridization. To study the in vivo function of cflara, we generated a cflara knockout mutant zebrafish using transcription activator-like effector nucleases (TALENs). Frame shift mutation is caused by a 10-bp deletion in the first DED domain. By inbreeding the F1 generation, a homozygous mutant fish was produced and confirmed by PCR. Knockout of cflara leads to abnormal heart development and embryonic lethality in mice. However, mutant zebrafish did not show any differences from wild type in heartbeat rate, survival rate or development. Zebrafish have analogues of both cflara and cflarb. Quantitative PCR showed that cflarb mRNA levels of mutant zebrafish were higher than those in the wild type. In a chemical exposure experiment, mutant zebrafish larvae showed a longer survival rate compared with wild type after CoCl2 treatment. However, no significant difference was observed from cisplatin treatment. This data suggests that cflarb may contribute to normal development and causes a difference in chemical resistance.

MiR-24-3p regulates bladder cancer cell proliferation, migration, invasion and autophagy by targeting DEDD.

microRNAs (miRNAs), a class of small non-coding RNA molecules, can regulate gene expression by interacting with the 3'-untranslated regions (3'UTR) of target genes and influence various biological processes. We investigated the potential role of miR-24-3p in the development of bladder cancer by regulating DEDD, a member of the death effector domain-containing protein family. First, we found that miR-24-3p was highly expressed and that DEDD was expressed at a low level in bladder cancer tissues compared with that in adjacent bladder tissues by qRT-PCR (P<0.0001). Second, we found that miR-24-3p promoted the proliferation ability of bladder cancer cells using the MTT assay and colony forming assay; and showed that miR-24-3p accelerated the migration and invasion of bladder cancer cells using migration and invasion assays (P<0.05). Moreover, miR-24-3p inhibited apoptosis of bladder cancer cells, as shown by flow cytometry (P<0.05). Western blot results demonstrated that miR-24-3p participated in autophagy of bladder cancer cells by DEDD. In addition, the tumor formation assay showed that miR-24-3p promoted the growth of bladder tumor invivo. Furthermore, the luciferase reporter gene assay indicated that miR-24-3p suppressed DEDD gene transcription. Therefore, our study indicated that miR-24-3p promoted bladder cancer progression by inhibiting DEDD.

Influenza virus infection induces translocation of apoptosis-inducing factor (AIF) in A549 cells: role of AIF in apoptosis and viral propagation.

It is recognized that influenza virus induces caspase-dependent apoptosis by activating caspase-3. Apoptosis-inducing factor (AIF) is a caspase-independent cell death effector, and its mitochondrial-nuclear translocation plays an important role in apoptosis. It is demonstrated in this study how influenza virus infection can induce caspase-independent apoptosis in the human alveolar epithelial cell line A549. AIF is translocated from the mitochondria to the nucleus in a caspase-independent manner in response to infection with influenza virus. Knockdown of AIF expression by small interfering RNA (siRNA) led to a reduction in virus-infection-induced apoptosis and virus yield. These results indicate that AIF translocation has a role in influenza-virus-induced apoptosis.

Design and experimental validation of small-molecule inhibitors of the FADD protein

CD95 is one of the best-studied members of the death receptor family. Activation of CD95 leads to the induction of the cell death program, apoptosis, via formation of the death-inducing signaling complex (DISC). The Fas-Associated Death Domain (FADD) is a key adaptor protein in the formation of the CD95 DISC and activation of procaspase 8 in the receptor complex. FADD comprises the death domain and the death effector domain (DED). The death domain is needed for the interactions of FADD with CD95, while DED is necessary for the recruitment of procaspases 8 and 10 and the cFLIP protein into the DISC. The search for specific inhibitors that would prevent the interactions of FADD with other core proteins of the DISC is important for the studies of the structure and function of this complex, investigation of apoptosis mechanisms, and development of new treatments for neurodegenerative diseases. In the course of this work, an in silico screening for low-molecular-weight inhibitors that would selectively interact with FADD DED was performed. This work required the molecular modeling of the protein complexes and virtual screening of potential FADD inhibitors. In addition, a new technology to test the activity of these inhibitors was developed. The computational and experimental analysis allowed us to characterize the optimum conformation of FADD for the design of small molecules that would bind to the region of the amino acid residue F25. We presume that further optimization of the structures of the chemical compounds that can bind within the hydrophobic pocket next to F25 of FADD will allow the development of new candidate inhibitors of programmed cell death.

Tumor-targeted delivery of a C-terminally truncated FADD (N-FADD) significantly suppresses the B16F10 melanoma via enhancing apoptosis

Fas-associated protein with death domain (FADD), a pivotal adaptor protein transmitting apoptotic signals, is indispensable for the induction of extrinsic apoptosis. However, overexpression of FADD can form large, filamentous aggregates, termed death effector filaments (DEFs) by self-association and initiate apoptosis independent of receptor cross-linking. A mutant of FADD, which is truncated of the C-terminal tail (m-FADD, 182–205 aa) named N-FADD (m-FADD, 1–181 aa), can dramatically up-regulate the strength of FADD self-association and increase apoptosis. In this study, it was found that over-expression of FADD or N-FADD caused apoptosis of B16F10 cells in vitro, even more, N-FADD showed a more potent apoptotic effect than FADD. Meanwhile, Attenuated Salmonella Typhimurium strain VNP20009 was engineered to express FADD or N-FADD under the control of a hypoxia-induced NirB promoter and each named VNP-pN-FADD and VNP-pN-N-FADD. The results showed both VNP-pN-FADD and VNP-pN-N-FADD delayed tumor growth in B16F10 mice model, while VNP-pN-N-FADD suppressed melanoma growth more significantly than VNP-pN-FADD. Additionally, VNP-pN-FADD and VNP-pN-N-FADD induced apoptosis of tumor cells by activating caspase-dependent apoptotic pathway. Our results show that N-FADD is a more potent apoptotic inducer and VNP20009-mediated targeted expression of N-FADD provides a possible cancer gene therapeutic approach for the treatment of melanoma.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death.

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

Molecular and functional characterization of caspase-8 from the big-belly seahorse (Hippocampus abdominalis)

Apoptosis is a physiological process that can also participate in host immune defense mechanisms, including tumor growth suppression along with homeostasis and maturation of immune cells. Caspases are known to be involved in cellular apoptotic signaling; among them, caspase-8 plays an important role in the initiation phase of the apoptotic death cascade. In the current study, we molecularly characterized a caspase-8 homolog (designated as HaCasp-8) from Hippocampus abdominalis. The HaCasp-8 gene harbors a 1476 bp open reading frame (ORF) that codes for a protein of 492 amino acids (aa) with a predicted molecular mass of 55 kDa. HaCasp-8 houses the typical domain architecture of known initiator caspases, including the death effector domain and the carboxyl-terminal catalytic domain. As expected, phylogenetic analysis reflected a closer evolutionary relationship of HaCasp-8 with its teleostean similitudes. The results of our qPCR assays confirmed the ubiquitous expression of HaCasp-8 in physiologically important tissues examined, with pronounced expression levels in ovary tissues, followed by blood cells. HaCasp-8 expression at the mRNA level was found to be significantly modulated by lipopolysaccharide, polyinosinic:polycytidylic acid, Streptococcus iniae, and Edwardsiella tarda injection. Overexpression of HaCasp-8 could trigger a significant level of cell death in HEK293T cells, suggesting its putative role in cell death. Taken together, our findings suggest that HaCasp-8 is an important component in the caspase cascade, and its expression can be significantly modulated under pathogen stress conditions in the big-belly seahorse.

TIPE2 (Tumor Necrosis Factor α-induced Protein 8-like 2) Is a Novel Negative Regulator of TAK1 Signal

TIPE2 (TNF-α-induced protein 8-like 2) is a novel death effector domain protein and is a negative regulator of the innate and adaptive immune response. Although it has been demonstrated that caspase-8 contributes to the negative regulation of TIPE2, the negative regulatory mechanism is not entirely understood. Here, we demonstrate that TIPE2 interacts with TGF-β-activated kinase 1 (TAK1), a crucial regulatory molecule of inflammatory and immune signals, and consequently acts as a powerful negative regulator of TAK1. The interaction between endogenous TIPE2 and TAK1 was observed in RAW264.7 macrophage-like cells and mouse primary cells derived from spleen and thymus. The TIPE2 amino acid 101–140 region interacted with TAK1 by binding to the amino acid 200–291 region of the internal kinase domain of TAK1. TIPE2 interfered with the formation of the TAK1-TAB1-TAB2 complex and subsequently inhibited activation of TAK1 and its downstream molecules. Importantly, silencing TIPE2 through RNA interference attenuated the inhibitory action of TIPE2 on LPS- and TNF-α-stimulated TAK1 activity. Exogenous TIPE2 101–140, the region that interacts with TAK1, also inhibited LPS- and TNF-α-stimulated NF-κB reporter activity. Interestingly, cell-permeable TIPE2 protein maintained its binding ability with TAK1 and exhibited the same inhibitory action of native TIPE2 on TLR4 signaling in vitro. Thus, cell-permeable TIPE2 protein is a potential candidate for intracellular protein therapy for TAK1-related diseases. The present study demonstrates that TIPE2 acts as a novel negative regulator of inflammatory and immune responses through TAK1 signaling.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation invitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.

Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae.

Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX-mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re-sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae.

When a Stomach Bug Comes Calling

They say that an army marches on its stomach, and while history provides ample illustration of the value of robust supply lines, the quality of the stores being delivered matters equally. In our modern world (where a growing population is truly an army writ large), food safety is of paramount importance for human health and economic stability. Viral and bacterial gastrointestinal infections transmitted from individual to individual during food preparation or from compromised food sources directly can be acutely debilitating and even fatal for subsets of the population, including young children.

ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces

Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASC·ASC and ASC·NLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling.

Cell death triggering and effector recognition by Sw-5 SD-CNL proteins from resistant and susceptible tomato isolines to Tomato spotted wilt virus.

SummaryOnly a limited number of dominant resistance genes acting against plant viruses have been cloned, and further functional studies of these have been almost entirely limited to the resistance genes Rx against Potato virus X (PVX) and N against Tobacco mosaic virus (TMV). Recently, the cell‐to‐cell movement protein (NSM) of Tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant (Avr) of Sw‐5b‐mediated resistance, a dominant resistance gene which belongs to the class of SD‐CC‐NB‐LRR (Solanaceae domain‐coiled coil‐nucleotide‐binding‐leucine‐rich repeat, SD‐CNL) resistance genes. On transient expression of the NSM protein in tomato and transgenic Nicotiana benthamiana harbouring the Sw‐5b gene, a hypersensitive cell death response (HR) is triggered. Here, it is shown that high accumulation of the Sw‐5b protein in N. benthamiana leaves, achieved by co‐expression of the Sw‐5b protein with RNA silencing suppressors (RSSs), leads to auto‐activity in the absence of NSM. In a similar approach, Sw‐5a, the highest conserved paralogue of Sw‐5b from Solanum peruvianum, also triggered HR by auto‐activation, whereas the highest conserved orthologue from susceptible S. lycopersicum, named Sw‐5aS, did not. However, neither of the last two homologues was able to trigger an NSM‐dependent HR. Truncated and mutated versions of these Sw‐5 proteins revealed that the NB‐ARC [nucleotide‐binding adaptor shared by Apaf‐1 (from humans), R proteins and CED‐4 (from nematodes)] domain is sufficient for the triggering of HR and seems to be suppressed by the SD‐CC domain. Furthermore, a single mutation was sufficient to restore auto‐activity within the NB‐ARC domain of Sw‐5aS. When the latter domain was fused to the Sw‐5b LRR domain, NSM‐dependent HR triggering was regained, but not in the presence of its own Sw‐5aS LRR domain. Expression analysis in planta revealed a nucleocytoplasmic localization pattern of Sw‐5b, in which the SD‐CC domain seems to be required for nuclear translocation. Although the Sw‐5 N‐terminal CC domain, in contrast with Rx, contains an additional SD, most findings from this study support a conserved role of domains within NB‐LRR (NLR) proteins against plant viruses.

The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress.

Hypoxia is a major hallmark of the tumor microenvironment that is strictly associated with rapid cancer progression and induction of metastasis. Hypoxia inhibits disulfide bond formation and impairs protein folding in the Endoplasmic Reticulum (ER). The stress in the ER induces the activation of Unfolded Protein Response (UPR) pathways via the induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, the level of phosphorylated Eukaryotic Initiation Factor 2 alpha (eIF2α) is markedly elevated, resulting in the promotion of a pro-adaptive signaling pathway by the inhibition of global protein synthesis and selective translation of Activating Transcription Factor 4 (ATF4). On the contrary, during conditions of prolonged ER stress, pro-adaptive responses fail and apoptotic cell death ensues. Interestingly, similar to the activity of the mitochondria, the ER may also directly activate the apoptotic pathway through ER stress-mediated leakage of calcium into the cytoplasm that leads to the activation of death effectors. Apoptotic cell death also ensues by ATF4-CHOP- mediated induction of several pro-apoptotic genes and suppression of the synthesis of anti-apoptotic Bcl-2 proteins. Advancing molecular insight into the transition of tumor cells from adaptation to apoptosis under hypoxia-induced ER stress may provide answers on how to overcome the limitations of current anti-tumor therapies. Targeting components of the UPR pathways may provide more effective elimination of tumor cells and as a result, contribute to the development of more promising anti-tumor therapeutic agents.

Abstract 2459: Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering

Abstract Development of multidrug resistance poses a persistent problem in cancer treatment, yet its underlying causes remain obscure. A principal role for mitochondria has been sought as this organelle integrates diverse stress signals to impact cell fate. Endoplasmic reticulum (ER) and mitochondria (mito) interact at specialized coupling sites called mitochondria-associated ER membranes (MAMs). MAMs serve as micro-domains for the transfer of essential calcium and lipid signals to mitochondria and regulate apoptotic sensitivity. Tightening of ER-mito tethering constitutes an early response to cellular stress leading to apoptosis, and alterations in ER-mito tethering have been implicated in diabetes and neurodegeneration, suggesting that the deregulation of this process may have broad relevance in disease. Here, we define a novel mechanism for chemotherapy resistance due to selection for reduced ER-mito tethering. Most high-risk neuroblastoma patients initially respond to chemotherapy before relapsing with lethal therapy resistant disease acquired during the course of intensive multimodality treatment. We obtained isogenic neuroblastoma cell lines from the same 7 patients both at the time of diagnosis (chemosensitive) and at the time of relapse (chemoresistant). We evaluated mitochondrial biomass (citrate synthase activity), mtDNA content (qPCR), and mtDNA sequence (Affymetrix MitoChip v2.0) but identified no changes correlated with acquired resistance. Electron microscopy image analyses of ER-mito interfaces revealed that tumors at relapse contain up to 70% fewer ER-mito tether complexes than their matched at-diagnosis tumors, as confirmed by IB for organelle-specific proteins. Mitochondria isolated from all 7 post-relapse tumors show attenuated cytochrome c release in response to tBid and Bim BH3 peptide, terminal death effectors downstream of most therapeutic stress. This attenuated mitochondrial response can be phenocopied by limited protelolysis of mitochondria to reduce ER-mito tethers. Reduced mitochondrial apoptotic signaling in post-relapse tumors correlates directly with chemoresistance (up to 800-fold) across diverse agent classes. To functionally validate this relationship, Cyclosporine A (CsA), a cyclophilin D inhibitor, was used in tumors at diagnosis to reduce ER-mito tethering. This led to attenuated apoptotic responses in isolated mitochondria, and increased tumor cell IC50 to diverse chemotherapeutics, partially phenocopying the therapy resistant state. Our findings support a novel model of differential apoptotic signaling in therapy resistant cells that relies on the altered proximity and interactions of the mitochondria with ER that may be harnessed to design more effective anti-cancer drug therapies. Citation Format: Jorida Coku, Elizabeth O. Scadden, Kangning Liu, Annette Vu, David M. Booth, Michelle Chen, Sharon Kim, C. Patrick Reynolds, György Hajnóczky, Michael D. Hogarty. Chemotherapy resistance in pediatric neuroblastoma is associated with reduced ER-mitochondria tethering. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2459.

Correction: Simultaneous Induction of Non-Canonical Autophagy and Apoptosis in Cancer Cells by ROS-Dependent ERK and JNK Activation

[This corrects the article DOI: 10.1371/journal.pone.0009996.].