Cellular Inhibitor of Apoptosis (cIAP)-mediated ubiquitination of Phosphofurin Acidic Cluster Sorting protein 2 (PACS-2) negatively regulates Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) cytotoxicity

Lysosomal membrane permeabilization is an essential step in TRAIL-induced apoptosis of liver cancer cell lines. TRAIL-induced lysosomal membrane permeabilization is mediated by the multifunctional sorting protein PACS-2 and repressed by the E3 ligases cIAP-1 and cIAP-2. Despite the opposing roles for PACS-2 and cIAPs in TRAIL-induced apoptosis, an interaction between these proteins has yet to be examined. Herein, we report that cIAP-1 and cIAP-2 confer TRAIL resistance to hepatobiliary cancer cell lines by reducing PACS-2 levels. Under basal conditions, PACS-2 underwent K48-linked polyubiquitination, resulting in PACS-2 proteasomal degradation. Biochemical assays showed cIAP-1 and cIAP-2 interacted with PACS-2 in vitro and co-immunoprecipitation studies demonstrated that the two cIAPs bound PACS-2 in vivo. More importantly, both cIAP-1 and cIAP-2 directly mediated PACS-2 ubiquitination in a cell-free assay. Single c-Iap-1 or c-Iap-2 gene knock-outs in mouse hepatocytes did not lead to PACS-2 accumulation. However, deletion of both cIAP-1 and cIAP-2 reduced PACS-2 ubiquitination, which increased PACS-2 levels and sensitized HuH-7 cells to TRAIL-induced lysosomal membrane permeabilization and apoptosis. Correspondingly, deletion of cIAPs sensitized wild-type, but not PACS-2-deficient hepatocarcinoma cells or Pacs-2-/- mouse hepatocytes to TRAIL-induced apoptosis. Together, these data suggest cIAPs constitutively downregulate PACS-2 by polyubiquitination and proteasomal degradation, thereby restraining TRAIL-induced killing of liver cancer cells. © 2014 Guicciardi et al

Death receptor 5 internalization is required for lysosomal permeabilization by TRAIL in malignant liver cell lines.

BACKGROUND & AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity in hepatocellular carcinoma cells is mediated by lysosomal permeabilization. Our aims were to determine which TRAIL receptor, death receptor (DR) 4 or DR5, mediates lysosomal permeabilization and assess whether receptor endocytosis followed by trafficking to lysosomes contributes in this process. METHODS: TRAIL ligand internalization in Huh-7 cells was examined by confocal microscopy using Flag-tagged TRAIL, whereas DR4- and DR5-enhanced green fluorescent protein internalization was assessed by total internal reflection microscopy. Clathrin-dependent endocytosis was inhibited by expressing dominant negative dynamin. RESULTS: Although Huh-7 cells express both TRAIL receptors, short hairpin RNA silencing of DR5 but not DR4 attenuated TRAIL-mediated lysosomal permeabilization and apoptosis. The TRAIL/DR5 complex underwent rapid cellular internalization upon ligand stimulation, whereas the TRAIL/DR4 complex was not efficiently internalized. DR5-enhanced green fluorescent protein internalization was dependent on a dileucine-based internalization motif. Endocytosis of the TRAIL/DR5 complex was dynamin dependent and was required for rapid lysosomal permeabilization and apoptosis in multiple malignant hepatocellular and cholangiocarcinoma cell lines. Upon TRAIL treatment, DR5 colocalized with lysosomes after internalization. Inhibition of DR5 trafficking to lysosomes by Rab7 small interfering RNA also reduced TRAIL-mediated lysosomal disruption and apoptosis. CONCLUSIONS: TRAIL-mediated endocytosis of DR5 with trafficking to lysosomes contributes to lysosomal protease release into the cytosol and efficient apoptosis in malignant liver cell lines

Death receptor 5 signaling promotes hepatocyte lipoapoptosis.

Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor

Neuronal vulnerability and multilineage diversity in multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing-remitting disease course at early stages, distinct lesion characteristics in cortical grey versus subcortical white matter and neurodegeneration at chronic stages. Here we used single-nucleus RNA sequencing to assess changes in expression in multiple cell lineages in MS lesions and validated the results using multiplex in situ hybridization. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper-cortical layers underlying meningeal inflammation; such MS neuron populations exhibited upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated microglia mapped most strongly to the rim of MS plaques. Notably, single-nucleus RNA sequencing identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to progression of MS lesions.Includes NIHR, ERC and Wellcome Trust

Agonistic Interventions into Public Commemorative Art:An Innovative Form of Counter-memorial Practice?

In light of recent controversies around the removal or modification of public commemorative art, such as memorials and monuments, this paper interrogates the value of competing approaches to counter-memorial practice using the framework of agonistic memory. It argues that much counter-memorial practice today, as it relates to historical memory, is dominated by a “cosmopolitan” mode that fails to offer a convincing response to the rise of right-wing populism and its instrumentalization of conflicts over public commemorative art. The article investigates two case studies of counter-memorial interventions that focus on the memory of fascism in Europe today and seeks to identify and assess emergent agonistic practices

Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas

<p>Abstract</p> <p>Background</p> <p>Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC.</p> <p>Methods</p> <p>Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs.</p> <p>Results</p> <p>EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis.</p> <p>Conclusion</p> <p>These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.</p

Dysregulation of Macrophage-Secreted Cathepsin B Contributes to HIV-1-Linked Neuronal Apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1ADA infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages