Caspase-8-Dependent Inflammatory Responses Are Controlled by Its Adaptor, FADD, and Necroptosis.

Cell death pathways regulate various homeostatic processes. Autoimmune lymphoproliferative syndrome (ALPS) in humans and lymphoproliferative (LPR) disease in mice result from abrogated CD95-induced apoptosis. Because caspase-8 mediates CD95 signaling, we applied genetic approaches to dissect the roles of caspase-8 in cell death and inflammation. Here, we describe oligomerization-deficient Caspase-8F122GL123G/F122GL123G and non-cleavable Caspase-8D387A/D387A mutant mice with defective caspase-8-mediated apoptosis. Although neither mouse developed LPR disease, removal of the necroptosis effector Mlkl from Caspase-8D387A/D387A mice revealed an inflammatory role of caspase-8. Ablation of one allele of Fasl, Fadd, or Ripk1 prevented the pathology of Casp8D387A/D387AMlkl-/- animals. Removing both Fadd alleles from these mice resulted in early lethality prior to post-natal day 15 (P15), which was prevented by co-ablation of either Ripk1 or Caspase-1. Our results suggest an in vivo role of the inflammatory RIPK1-caspase-8-FADD (FADDosome) complex and reveal a FADD-independent inflammatory role of caspase-8 that involves activation of an inflammasome.

microRNA 125a Regulates MHC-I Expression on Esophageal Adenocarcinoma Cells, Associated With Suppression of Antitumor Immune Response and Poor Outcomes of Patients.

BACKGROUND & AIMS: Immune checkpoint inhibition may affect growth or progression of highly aggressive cancers, such as esophageal adenocarcinoma (EAC). We investigated the regulation of expression of major histocompatibility complex, class 1 (MHC-I) proteins (encoded by HLA-A, HLA-B, and HLA-C) and the immune response to EACs in patient samples. METHODS: We performed quantitative polymerase chain reaction array analyses of OE33 cells and OE19 cells, which express different levels of the ATP binding cassette subfamily B member 1 (TAP1) and TAP2, required for antigen presentation by MHC-I, to identify microRNAs (miRNAs) that regulate their expression. We performed luciferase assays to validate interactions between miRNAs and potential targets. We overexpressed candidate miRNAs in OE33, FLO-1, and OACP4 C cell lines and performed quantitative polymerase chain reaction, immunoblot, and flow cytometry analyses to identify changes in messenger RNA (mRNA) and protein expression; we studied the effects of cytotoxic T cells. We performed miRNA in situ hybridization, RNA-sequencing, and immunohistochemical analyses of tumor tissues from 51 untreated patients with EAC in the Netherlands. Clinical and survival data were collected for patients, and EAC subtypes were determined. RESULTS: We found OE19 cells to have increased levels of 7 miRNAs. Of these, we found binding sites for miRNA 125a (MIR125a)-5p in the 3' untranslated region of the TAP2 mRNA and binding sites for MIR148a-3p in 3' untranslated regions of HLA-A, HLA-B, and HLA-C mRNAs. Overexpression of these miRNAs reduced expression of TAP2 in OE33, FLO-1, and OACP4 C cells, and reduced cell-surface levels of MHC-I. OE33 cells that expressed the viral peptide BZLF1 were killed by cytotoxic T cells, whereas OE33 that overexpressed MIR125a-5p or MIR 148a along with BZLF1 were not. In EAC and nontumor tissues, levels of MIR125a-5p correlated inversely with levels of TAP2 protein. High expression of TAP1 by EAC correlated with significantly shorter overall survival times of patients. EACs that expressed high levels of TAP1 and genes involved in antigen presentation also expressed high levels of genes that regulate the adaptive immune response, PD-L1, PD-L2, and IDO1; these EACs had a poor response to neoadjuvant chemoradiotherapy and associated with shorter overall survival times of patients. CONCLUSIONS: In studies of EAC cell lines and tumor tissues, we found increased levels of MIR125a-5p and MIR148a-3p to reduce levels of TAP2 and MHC-I, required for antigen presentation. High expression of MHC-I molecules by EAC correlated with markers of an adaptive immune response and significantly shorter overall survival times of patients.

Mitophagy restricts BAX/BAK-independent, Parkin-mediated apoptosis.

Degradation of defective mitochondria is an essential process to maintain cellular homeostasis and it is strictly regulated by the ubiquitin-proteasome system (UPS) and lysosomal activities. Here, using genome-wide CRISPR and small interference RNA screens, we identified a critical contribution of the lysosomal system in controlling aberrant induction of apoptosis following mitochondrial damage. After treatment with mitochondrial toxins, activation of the PINK1-Parkin axis triggered a BAX- and BAK-independent process of cytochrome c release from mitochondria followed by APAF1 and caspase 9-dependent apoptosis. This phenomenon was mediated by UPS-dependent outer mitochondrial membrane (OMM) degradation and was reversed using proteasome inhibitors. We found that the subsequent recruitment of the autophagy machinery to the OMM protected cells from apoptosis, mediating the lysosomal degradation of dysfunctional mitochondria. Our results underscore a major role of the autophagy machinery in counteracting aberrant noncanonical apoptosis and identified autophagy receptors as key elements in the regulation of this process.

GM1 Oligosaccharide Efficacy in Parkinson's Disease: Protection against MPTP.

Past evidence has shown that the exogenous administration of GM1 ganglioside slowed neuronal death in preclinical models of Parkinson's disease, a neurodegenerative disorder characterized by the progressive loss of dopamine-producing neurons: however, the physical and chemical properties of GM1 (i.e., amphiphilicity) limited its clinical application, as the crossing of the blood-brain barrier is denied. Recently, we demonstrated that the GM1 oligosaccharide head group (GM1-OS) is the GM1 bioactive portion that, interacting with the TrkA-NGF complex at the membrane surface, promotes the activation of a multivariate network of intracellular events regulating neuronal differentiation, protection, and reparation. Here, we evaluated the GM1-OS neuroprotective potential against the Parkinson's disease-linked neurotoxin MPTP, which destroys dopaminergic neurons by affecting mitochondrial bioenergetics and causing ROS overproduction. In dopaminergic and glutamatergic primary cultures, GM1-OS administration significantly increased neuronal survival, preserved neurite network, and reduced mitochondrial ROS production enhancing the mTOR/Akt/GSK3ß pathway. These data highlight the neuroprotective efficacy of GM1-OS in parkinsonian models through the implementation of mitochondrial function and reduction in oxidative stress.

GM1 oligosaccharide efficacy against α-synuclein aggregation and toxicity in vitro.

Fibrillary aggregated α-synuclein represents the neurologic hallmark of Parkinson's disease and is considered to play a causative role in the disease. Although the causes leading to α-synuclein aggregation are not clear, the GM1 ganglioside interaction is recognized to prevent this process. How GM1 exerts these functions is not completely clear, although a primary role of its soluble oligosaccharide (GM1-OS) is emerging. Indeed, we recently identified GM1-OS as the bioactive moiety responsible for GM1 neurotrophic and neuroprotective properties, specifically reverting the parkinsonian phenotype both in in vitro and in vivo models. Here, we report on GM1-OS efficacy against the α-synuclein aggregation and toxicity in vitro. By amyloid seeding aggregation assay and NMR spectroscopy, we demonstrated that GM1-OS was able to prevent both the spontaneous and the prion-like α-synuclein aggregation. Additionally, circular dichroism spectroscopy of recombinant monomeric α-synuclein showed that GM1-OS did not induce any change in α-synuclein secondary structure. Importantly, GM1-OS significantly increased neuronal survival and preserved neurite networks of dopaminergic neurons affected by α-synuclein oligomers, together with a reduction of microglia activation. These data further demonstrate that the ganglioside GM1 acts through its oligosaccharide also in preventing the α-synuclein pathogenic aggregation in Parkinson's disease, opening a perspective window for GM1-OS as drug candidate.

GM1 ganglioside exerts protective effects against glutamate-excitotoxicity via its oligosaccharide in wild-type and amyotrophic lateral sclerosis motor neurons.

Alterations in glycosphingolipid metabolism have been linked to the pathophysiological mechanisms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons. Accordingly, administration of GM1, a sialic acid-containing glycosphingolipid, is protective against neuronal damage and supports neuronal homeostasis, with these effects mediated by its bioactive component, the oligosaccharide head (GM1-OS). Here, we add new evidence to the therapeutic efficacy of GM1 in ALS: Its administration to WT and SOD1G93A motor neurons affected by glutamate-induced excitotoxicity significantly increased neuronal survival and preserved neurite networks, counteracting intracellular protein accumulation and mitochondria impairment. Importantly, the GM1-OS faithfully replicates GM1 activity, emphasizing that even in ALS the protective function of GM1 strictly depends on its pentasaccharide.

Immune signaling and regulation in esophageal cancer.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on signaling pathways that can be targeted with immunotherapy; the role of micro-RNAs in the immune response to esophageal cancer; and the association between obesity, the immune system, and esophageal adenocarcinoma.

A pSMAD/CDX2 complex is essential for the intestinalization of epithelial metaplasia.

The molecular mechanisms leading to epithelial metaplasias are poorly understood. Barrett's esophagus is a premalignant metaplastic change of the esophageal epithelium into columnar epithelium, occurring in patients suffering from gastroesophageal reflux disease. Mechanisms behind the development of the intestinal subtype, which is associated with the highest cancer risk, are unclear. In humans, it has been suggested that a nonspecialized columnar metaplasia precedes the development of intestinal metaplasia. Here, we propose that a complex made up of at least two factors needs to be activated simultaneously to drive the expression of intestinal type of genes. Using unique animal models and robust in vitro assays, we show that the nonspecialized columnar metaplasia is a precursor of intestinal metaplasia and that pSMAD/CDX2 interaction is essential for the switch toward an intestinal phenotype.

Nano-curcumin inhibits proliferation of esophageal adenocarcinoma cells and enhances the T cell mediated immune response.

In Western countries the incidence of the esophageal adenocarcinoma (EAC) has risen at a more rapid rate than that of any other malignancy. Despite intensive therapies this cancer is associated with extreme high morbidity and mortality. For this reason, novel effective therapeutic strategies are urgently required. Dendritic Cell (DC)-based immunotherapy is a promising novel treatment strategy, which combined with other anti-cancer strategies has been proven to be beneficial for cancer patients. Curcumin (diferuloylmethane), is a natural polyphenol that is known for its anti-cancer effects however, in it's free form, curcumin has poor bioavailability. The aim of this study was to investigate whether using a highly absorptive form of curcumin, dispersed with colloidal nano-particles, named Theracurmin would be more effective against EAC cells and to analyze if this new compound affects DC-induced T cell response. As a result, we show efficient uptake of nano-curcumin by the EAC cell lines, OE33, and OE19. Moreover, nano-curcumin significantly decreased the proliferation of the EAC cells, while did not affect the normal esophageal cell line HET-1A. We also found that nano-curcumin significantly up-regulated the expression of the co-stimulatory molecule CD86 in DCs and significantly decreased the secretion of pro-inflammatory cytokines from in vitro activated T cells. When we combined T cells with nano-curcumin treatment in OE19 and OE33, we found that the basic levels of T cell induced cytotoxicity of 6.4 and 4.1%, increased to 15 and 13%, respectively. In conclusion, we found that nano-curcumin is effective against EAC, sensitizes EAC cells to T cell induced cytotoxicity and decreases the pro-inflammatory signals from T cells. Combining DC immunotherapy with nano-curcumin is potentially a promising approach for future treatment of EAC.