Loss Of Caspase Research Articles

ZBP1 and TRIF trigger lethal necroptosis in mice lacking caspase-8 and TNFR1.

Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues. RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many barrier epithelia. ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl. Intriguingly, expression of ZBP1 was elevated in Casp8-/- Tnfr1-/- embryos prior to their succumbing to aberrant necroptosis around embryonic day 15 (E15). ZBP1 contributed to this embryonic lethality because rare Casp8-/- Tnfr1-/- Zbp1-/- mice survived until after birth. Necroptosis mediated by TRIF contributed to the demise of Casp8-/- Tnfr1-/- Zbp1-/- pups in the perinatal period. Of note, Casp8-/- Tnfr1-/- Trif-/- Zbp1-/- mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8-/- Ripk1-/- Trif-/- Zbp1-/-, Casp8-/- Ripk3-/-, or Casp8-/- Mlkl-/- mice. Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.

RIPK3 and caspase 8 collaborate to limit herpes simplex encephalitis.

Invasion of the brain by herpes simplex virus 1 (HSV1) can lead to the development of herpes simplex encephalitis (HSE) that is often associated with significant morbidity and mortality regardless of therapeutic intervention. Both virus and host immune factors dictate HSE onset and progression. Because programmed cell death pathways including necroptosis are important antiviral defense mechanisms in HSV1-associated peripheral diseases, they might also play critical roles in HSV1 neuropathogenesis. HSV1-encoded ICP6 prevents receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis during infection of human cells, but it also acts as a species-dependent inducer of necroptosis in murine cells and thereby restricts virus replication. We therefore used an established mouse model of HSE to investigate RIPK3-mediated necroptosis impact on HSV1 neuropathogenesis. Following corneal HSV1 inoculation, RIPK3 knockout mice showed increased susceptibility to HSE when compared with wildtype mice indicating RIPK3 helps to limit HSE progression. RIPK3-mediated defense against HSE was found to be independent of the kinase domain necessary to drive necroptosis implicating that a death independent function of RIPK3 protects against HSE. Conversely the pro-necroptotic kinase function RIPK3 served to limit viral replication in corneal tissue implicating a tissue-specific RIPK3 function in limiting HSV1. Further evaluation of the kinase-independent mechanism to restrict HSE revealed that the RIPK3 signaling partner, caspase 8, contributes to limiting HSE neuropathogenesis. Increased HSE susceptibility from loss of caspase 8 and RIPK3 correlated with decreased levels of chemokines, cytokines, and antiviral lymphocytes recruitment to the brain. We conclude that RIPK3 contributes toward host control of HSV1 replication in a tissue-specific fashion. Whereas RIPK3-mediated necroptosis restricts virus replication within the cornea, kinase-independent induction of inflammation by RIPK3 in collaboration with caspase 8 restricts virus replication within the brain during HSE neuropathogenesis.

LUBAC suppresses IL-21-induced apoptosis in CD40-activated murine B cells and promotes germinal center B cell survival and the T-dependent antibody response

Abstract B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-kB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown here, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm) could undergo proliferation, CSR and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a high-affinity BCR mutant and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-kB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and -independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors, but virtually abrogated – together with Linear M1-Ub and cFLIP levels – by a small molecule HOIP inhibitor. Thus, LUBAC controls cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.

Caspase-8 loss radiosensitizes head and neck squamous cell carcinoma to SMAC mimetic-induced necroptosis.

Caspase-8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCCs), and CASP8 mutations are associated with poor survival. The distribution of these mutations in HNSCCs suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a regulated cell death mechanism. Here, we show that knockdown of CASP8 renders HNSCCs susceptible to necroptosis by a second mitochondria-derived activator of caspase (SMAC) mimetic, birinapant, in combination with pan-caspase inhibitors Z-VAD-FMK or emricasan and radiation. In a syngeneic mouse model of oral cancer, birinapant, particularly when combined with radiation, delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine protein kinase 3 (RIP3) determines susceptibility to this mode of death. Although an in vitro screen revealed that low RIP3 levels rendered many HNSCC cell lines resistant to necroptosis, patient tumors maintained RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be relevant therapeutically in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained.

Mitochondrial ROS prime the hyperglycemic shift from apoptosis to necroptosis

We have previously identified a shift from TNF-α-induced apoptosis to necroptosis that occurs under hyperglycemic conditions. This shift involves the downregulation or silencing of caspases and concurrent upregulation of necroptotic proteins leading to activation of the necrosome. In addition, under hyperglycemic conditions in vivo, this shift in cell death mechanisms exacerbates neonatal hypoxia-ischemia (HI) brain injury. Here, we identify two major factors that drive the hyperglycemic shift to necroptosis: (1) reactive oxygen species (ROS) and (2) receptor-interacting protein kinase 1 (RIP1). ROS, including mitochondrial superoxide, led to the oxidation of RIP1, as well as formation and activation of the necrosome. Concurrently, ROS mediate a decrease in the levels and activation of executioner caspases-3, -6, and -7. Importantly, hyperglycemia and mitochondrial ROS result in the oxidation of RIP1 and loss of executioner caspases prior to death receptor engagement by TNF-α. Moreover, RIP1 partially controlled levels of mitochondrial ROS in the context of hyperglycemia. As a result of its regulation of ROS, RIP1 also regulated necrosome activation and caspase loss. Mitochondrial ROS exacerbated neonatal HI-brain injury in hyperglycemic mice, as a result of the shift from apoptosis to necroptosis.

Abstract B18: Loss of caspase-8 renders head and neck squamous cell carcinomas susceptible to necroptosis

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, with 500,000 new diagnoses and 300,000 cancer-related deaths worldwide annually. Despite 50 years of basic, clinical, and translational research, survival rates for patients with HNSCC have not remarkably improved, indicating a need for identification of new targets and biomarkers to guide therapy. Recent sequencing data from our group and others have identified Caspase-8 (CASP8) as one of the most frequently mutated genes in HNSCCs, with CASP8 mutations occurring in ~10% of the primary tumors. Genomics analysis using The Cancer Genome Atlas (TCGA) HNSCC dataset revealed that CASP8 mutations are associated with poorer overall patient survival. CASP8 belongs to the caspase family of proteases and has important roles in both apoptotic and necroptotic death. Our goal was to understand CASP8 signaling in HNSCC and examine whether those pathways could be exploited therapeutically. CASP8 serves as the apical-most caspase for the extrinsic apoptosis pathway, initiating programmed cell death following death receptor ligation. Cells deficient in Caspase-8 show resistance to death-receptor mediated killing in vitro and in vivo. Intriguingly, however, knockout of CASP8 in some tissues promotes cell death through induction of necroptosis, a.k.a programmed necrosis. Necroptosis is mediated by receptor-interacting protein kinases RIP1, RIP3 and their substrate mixed lineage kinase-like (MLKL) and often occurs following activation of tumor necrosis factor alpha (TNF-alpha) signaling. CASP8 has been shown to cleave and inhibit RIP1 and RIP3, thereby suppressing necroptosis. We hypothesized that mutation or loss of CASP8 could promote necroptosis and modulation of the pathway with small-molecule agents might have the potential for clinical utility. We found that deletion of CASP8 rendered HNSCCs susceptible to necroptosis induced by second mitochondria-derived activator of caspase (SMAC) mimetic treatment (TL-32711) when combined with TNF-alpha and Z-VAD-FMK, a pancaspase inhibitor, in vitro. Inhibition of Caspase-8 function was also associated with enhanced necroptotic killing by ionizing radiation in combination with SMAC mimetic treatment in vitro. We further demonstrated that the level of RIP3 expression determines susceptibility to SMAC mimetic-induced necroptosis in HNSCCs. In conclusion, CASP8 is an important regulator of cell death pathways in HNSCC. Modulation of these pathways can enhance the efficacy of radiotherapy and has the potential for clinical translation. Ongoing studies will give further insight into how CASP8 regulates necroptosis in HNSCCs and provide preclinical justification for use of these pathways as a therapeutic target in HNSCC patients. Citation Format: Burak Uzunparmak, Jeffrey N. Myers, Curtis R. Pickering. Loss of caspase-8 renders head and neck squamous cell carcinomas susceptible to necroptosis [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B18.

708P - A phase I/Ib, multi-center trial of ARQ-761 (Beta-Lapachone) with gemcitabine/nab-paclitaxel in patients with advanced pancreatic cancer

BackgroundARQ761 (β-lapachone) is a hydroquinone analog that exploits the unique elevation of NQO1 found in 90% of pancreatic cancer cells and causes tumor-specific cell death by eliciting a futile redox cycle generating high levels of reactive oxygen species and ultimately PARP1 hyperactivation-dependent cell death. ARQ761 induces cell death which is: (i) not dependent on p53 status; (ii) not dependent on cell cycle status; (iii) not affected by known oncogenic driver or carrier mutations; and (iv) not affected by loss of caspases. We demonstrated that ARQ761 has synergistic antitumor activity with chemotherapy in pancreatic cancer preclinical models. MethodsThis was a single arm phase-1b clinical trial of ARQ-761 plus gemcitabine and nab-paclitaxel for patients with pancreatic cancer. The primary objective was to determine the maximum tolerated dose of ARQ 761 in combination with chemotherapy. Secondary objectives were to assess clinical activity, safety, tolerability and pharmacodynamic effects. Patients with metastatic pancreatic adenocarcinoma not treated with prior gemcitabine with adequate hematologic, renal and liver function and good performance status were enrolled. Dose levels are described in the table. Immunohistochemistry was used to assess for NQO1 expression. ResultsFifteen patients were evaluable for safety, 67% were male, 53% had received prior chemotherapy and 96% tested positive for NQO1 expression. The most common treatment related toxicities were anemia and fatigue. The following dose limiting toxicities were observed (dose level 1: neutropenia, anemia, dose level -1 fatigue). The maximum tolerated dose of ARQ761/gemcitabine/nab-paclitaxel is 156/800/100mg/m2. Tumour response was stable disease in 53% Pharmacodynamic analysis was performed on post treatment biopsies. ConclusionsWe report results of a phase 1b clinical trial of the novel NQO1 bioactivatable compound, ARQ761, in combination with gemcitabine/nab-paclitaxel. Treatment related toxicities were observed which were noted in preclinical models and managed with routine dose adjustment. Clinical trial identificationNCT02514031. Legal entity responsible for the studyUT Southwestern Medical Center. FundingPancreatic Cancer Action Network, Rising Tide, Gateway, Arqule Pharmaceuticals. DisclosureM.S. Beg: Research grant / Funding (institution): Arqule; Advisory / Consultancy: Array; Advisory / Consultancy, Speaker Bureau / Expert testimony: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: Ipsen; Advisory / Consultancy: Boston Biomedical; Advisory / Consultancy: Guardant; Advisory / Consultancy: Genentech. D. Boothman: Research grant / Funding (institution): Arqule; Advisory / Consultancy: Toray global; Speaker Bureau / Expert testimony: systems oncology. Y. Arriaga: Full / Part-time employment: IBM. A. Sanjeeviaiah: Advisory / Consultancy, 1500$: Guardant health. B. Schwartz: Full / Part-time employment: Arqule. All other authors have declared no conflicts of interest.

Inactivation of caspase 8 in liver parenchymal cells confers protection against murine obstructive cholestasis

Caspase 8 (CASP8) is the apical initiator caspase in death receptor-mediated apoptosis. Strong evidence for a link between death receptor signaling pathways and cholestasis has recently emerged. Herein, we investigated the role of CASP8-dependent and independent pathways during experimental cholestasis. Liver injury was characterized in a cohort of human sera (n = 28) and biopsies from patients with stage IV primary biliary cholangitis. In parallel, mice with either specific deletion of Casp8 in liver parenchymal cells (Casp8Δhepa) or hepatocytes (Casp8Δhep), and mice with constitutive Ripk3 (Ripk3-/-) deletion, were subjected to surgical ligation of the common bile duct (BDL) from 2 to 28 days. Floxed (Casp8fl/fl) and Ripk3+/+ mice were used as controls. Moreover, the pan-caspase inhibitor IDN-7314 was used, and cell death mechanisms were studied in primary isolated hepatocytes. Overexpression of activated caspase 3, CASP8 and RIPK3 was characteristic of liver explants from patients with primary biliary cholangitis. Twenty-eight days after BDL, Casp8Δhepamice showed decreased necrotic foci, serum aminotransferase levels and apoptosis along with diminished compensatory proliferation and ductular reaction. These results correlated with a decreased inflammatory profile and ameliorated liver fibrogenesis. A similar phenotype was observed in Ripk3-/- mice. IDN-7314 treatment decreased CASP8 levels but failed to prevent BDL-induced cholestasis, independently of CASP8 in hepatocytes. These findings show that intervention against CASP8 in liver parenchymal cells - specifically in cholangiocytes - might be a beneficial option for treating obstructive cholestasis, while broad pan-caspase inhibition might trigger undesirable side effects. Loss of caspase 8 - a protein involved in programmed cell death - in liver parenchymal cells protects against experimental cholestasis. Therefore, specific pharmacological intervention against caspase 8 might be a valid alternative for the treatment of obstructive cholestasis in the clinic, whereas broad pan-caspase inhibiting drugs might trigger undesirable side effects.

Notch inhibition counteracts Paneth cell death in absence of caspase-8.

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8∆int) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8∆int mice. The secretory cell metaplasia in DBZ-treated casp8∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Abstract AP26: SMAC–MIMETIC TREATMENT ENHANCES ANTITUMOR EFFECT OF STANDARD CHEMOTHERAPY IN OVARIAN CANCERS EXPRESSING LOW CASPASE 8 IN VITRO AND IN VIVO

Abstract Ovarian cancer is the most lethal gynecological cancer in the US. Standard treatment consists of surgery followed by chemotherapies relying on apoptotic tumor cell death. Most women with advanced stage disease will relapse, suggesting that this disease is characterized by primary and acquired resistance to chemotherapy, and targeted approaches to treatment are greatly needed. We recently demonstrated that low Caspase 8 expression levels in ovarian cancers correlated with resistance to chemotherapy, and a subpopulation of ovarian cancer patients with low Caspase 8 levels exhibits poorer overall survival after standard of care treatment. We hypothesized that the loss of Caspase 8 function reduces the cancer cells' ability to undergo apoptosis when exposed to standard chemotherapy. Here we show that combination treatment with Smac-mimetics targets tumor cells with low Caspase 8 and induces necroptotic cell death. We investigated the in vitro sequential effect of Smac-mimetic added to carboplatin + paclitaxel treatment of ovarian cancer cells expressing wild type, low and mutant Caspase 8 levels, which resulted in a 2-4fold enhancement of cell death. In vivo treatment of mice bearing subcutaneous or intraperitoneal ovarian xenografts showed greater aggressiveness of Caspase 8 deficient vs wild-type tumors; combined treatment with chemotherapy and Smac-mimetic resulted in over 50% decrease in low-Caspase 8 xenograft growth, as well as enhanced overall survival of the mice. Subpopulations of women with low Caspase 8 ovarian cancers that resist apoptosis could benefit from treatments which promote non-apoptotic forms of cell death, such as Smac-mimetic induced necroptosis. Clinical validation of such approaches are needed to increase the effectiveness of current standard ovarian cancer treatment. Citation Format: Lídia Hernandez and Christina M. Annunziata. SMAC–MIMETIC TREATMENT ENHANCES ANTITUMOR EFFECT OF STANDARD CHEMOTHERAPY IN OVARIAN CANCERS EXPRESSING LOW CASPASE 8 IN VITRO AND IN VIVO [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP26.

The Pseudokinase MLKL and the Kinase RIPK3 Have Distinct Roles in Autoimmune Disease Caused by Loss of Death-Receptor-Induced Apoptosis

The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease, and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice compared to Casp8(-/-)Ripk3(-/-) or Fadd(-/-)Ripk3(-/-) mice, respectively. These results demonstrate that MLKL is an essential effector of aberrant necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. Itappears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity, and excess cytokine production that occur when FADD or Caspase-8-mediated apoptosis is abrogated.

New insights into the regulation of innate immunity by caspase-8.

Caspase-8 is required for extrinsic apoptosis, but is also central for preventing a pro-inflammatory receptor interacting protein kinase (RIPK) 3–mixed lineage kinase domain-like (MLKL)-dependent cell death pathway termed necroptosis. Despite these critical cellular functions, the impact of capase-8 deletion in the myeloid cell lineage, which forms the basis for innate immune responses, has remained unclear. In a recent article in Arthritis Research & Therapy, Cuda et al. report that myeloid cell-restricted caspase-8 loss leads to a very mild RIPK3-dependent inflammatory phenotype. The presented results suggest that inflammation does not arise exclusively because of RIPK3-mediated necroptotic death but that, in the absence of caspase-8, RIPK1 and RIPK3 enhance microbiome-driven Toll-like receptor-induced pro-inflammatory cytokine production.

Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.

IntroductionAlthough caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population.MethodsCreLysMCasp8fl/fl mice were bred via a cross between Casp8fl/fl mice and CreLysM mice, and RIPK3−/−CreLysMCasp8fl/fl mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. CreLysMCasp8fl/fl mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann–Whitney U test.ResultsLoss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8–deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8–deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6ChighCD11b+F4/80+ splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in CreLysMCasp8fl/fl mice. Further, caspase-8–deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity.ConclusionsThese data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0794-z) contains supplementary material, which is available to authorized users.

Prognostic significance of TRAIL signalling molecules in cervical squamous cell carcinoma

AimTumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that preferentially induces apoptosis in cancer cells while exhibiting little or no toxicity in normal cells....

ET-10 * THE SYNERGISTIC EFFECT OF INHIBITOR OF INHIBITOR OF APOPTOSIS PROTEINS- LCL161 AND STANDARD ANTI-CANCER AGENTS ON MEDULLOBLASTOMA CELLS

Medulloblastoma is the most common malignant pediatric brain tumor, comprising 15-20% of intracranial tumor in children. Most metastatic and recurrent medulloblastoma cells are resistant to current therapeutic approaches including radiation and high-dose chemotherapy. In addition, some studies indicate that subsets of medulloblastomas exhibit loss of caspase-8 expression and therefore they can resist TRAIL-induced apoptosis.A chemo-drug, LCL161, has a mimic structure of Smac/DIABLO, which can inhibit XIAP and cIAP, and then enhance caspse-3, caspase-7 and caspase-9 activation. Our study purpose is to determine whether LCL161 induce cell apoptosis and activate caspases cascade through inhibition of IAPs in medulloblastoma cells(DAOY and d283) and to examine whether LCL161 can sensitize standard anti-cancer agents(vincrstine, or cisplatin) induced apoptosis. Based on prior data, DAOY cells had higher, and d283 cells had lower caspase-8 expressions respectively. LCL161 alone did not inhibit tumor growth for both medulloblastoma cells. Our data showed synergistic effect of LCL161-combined vincrstine, or cisplatin can be observed in DAOY cells. For d283 med cells, synergistic effect still exist but was not as significant as that of DAOY cells. LCL161-combined vincristine decreased around 40% and 20% cell viability in DAOY cells and d283 med cells compared to vincrisitne alone. LCL-combined cisplatin inhibited 20% growth rate in DAOY cells. Nevertheless, synergistic effect did not work in d283 cells. DNA flow and Annexin V/PI assay were indicated LCL 161 alone increased 16% of apoptotic cells. In addition, LCL161 enhanced 18% of apoptotic cells treated with vincristine, and the combined treatment approached to IC50 for medulloblastoma cells. LCL161-combined cisplatin increased 18% of apoptotic cells compared to cisplatin alone. Overall, the synergistic effect of LCL161 and standard anti-cancer agents was more significant on DAOY cells, which express higher caspase-8. The finding indicated that loss of caspase 8 play an important role in this therapeutic strategy.

Loss of caspase-8 in hepatocytes accelerates the onset of liver regeneration in mice through premature nuclear factor kappa B activation

The cytokine tumor necrosis factor alpha (TNF-α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF-κB) activation, apoptosis signaling by way of caspase-8 (Casp8), and activation of cJun N-terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8(Δhepa) ) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8(Δhepa) mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF-dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor-interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF-κB and JNK/cJun related signals. In order to define the role of NF-κB in this setting we blocked NF-κB activation in Casp8(Δhepa) mice by concomitant inactivation of the NF-κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8(Δhepa) mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF-κB, and JNK activation. While loss of Casp8 triggers NF-κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly.

Loss of Caspase 7 Expression Is Associated With Poor Prognosis in Renal Cell Carcinoma Clear Cell Subtype

To investigate the expression of CASP7 protein in renal cell carcinoma clear cell subtype (ccRCC) and its value to predict cancer-specific survival (CSS). A tissue microarray containing 120 samples of ccRCC, 45 non-ccRCC, and 66 nontumor paired samples from patients who underwent partial or radical nephrectomy was hybridized with anti-CASP7 antibody. Tissue sections were scored according to intensity and the percentage of stained cells. CASP7 immunostaining scores were used to estimate the association with clinicopathologic parameters and calculate Kaplan-Meier survival curves. Reduced CASP7 expression was observed in ccRCC and non-ccRCC subtypes in comparison with nontumor renal tissues (P<.0001). CASP7 immunostaining was associated (P<.05) with clinicopathologic parameters (size, incidental tumor, clinical stage, renal vein invasion, and tumor necrosis) and correlated with CSS (P= .032) and global survival (P= .046) of patients with ccRCC. In addition, CASP7 expression was able to substratify patients with ccRCC with favorable prognosis according to low clinical stage, in which negative CASP7 staining was associated with patients with lower CSS (P= .045). Finally, CASP7 staining was able to provide significant stratification according to CSS (P= .018) among patients with ccRCC with disease relapse. Our results implicate the loss of CASP7 expression in the aggressiveness of ccRCC and indicate its potential use as a clinical prognostic marker of the disease.

A role for c-FLIPL in the regulation of apoptosis, autophagy, and necroptosis in T lymphocytes

Caspase 8 plays a dual role in the survival of T lymphocytes. Although active caspase 8 mediates apoptosis upon death receptor signaling, the loss of caspase 8 activity leads to receptor-interacting protein (RIP)-1/RIP-3-dependent necrotic cell death (necroptosis) upon TCR activation. The anti-apoptotic protein c-FLIP (cellular caspase 8 (FLICE)-like inhibitory protein) suppresses death receptor-induced caspase 8 activation. Moreover, recent findings suggest that c-FLIP is also involved in inhibiting necroptosis and autophagy. It remains unclear whether c-FLIP protects primary T lymphocytes from necroptosis or regulates the threshold at which autophagy occurs. Here, we used a c-FLIP isoform-specific conditional deletion model to show that c-FLIP(L)-deficient T cells underwent RIP-1-dependent necroptosis upon TCR stimulation. Interestingly, although previous studies have only described necroptosis in the absence of caspase 8 activity, we found that pro-apoptotic caspase 8 activity and apoptosis were also enhanced in c-FLIP(L)-deficient T lymphocytes. Furthermore, c-FLIP(L)-deficient T cells exhibited enhanced autophagy, which served a cytoprotective function. Together, these findings indicate that c-FLIP(L) plays an important antinecroptotic role and is a key regulator of apoptosis, autophagy, and necroptosis in T lymphocytes.

Caspase-8 is essential for maintaining chromosomal stability and suppressing B-cell lymphomagenesis

In addition to its proapoptotic function, caspase-8 is also important for several other processes, including suppressing necroptosis, cell migration, and immune cell survival. In the present study, we report that the loss of caspase-8 in B lymphocytes leads to B-cell malignancies and that the risk for these tumors is further enhanced in the absence of p53. We also report that deficiency of caspase-8 results in impaired cytokinesis and that casp8(-/-) lymphomas display remarkably elevated levels of chromosomal aberrations. Our data support an important role for caspase-8 in the maintenance of genomic integrity and highlight its tumor-suppressive function.

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.