ABSTRACT
The prevalence of diabetes steadily increases worldwide mirroring the prevalence of obesity. Endoplasmic reticulum (ER) stress is activated in diabetes and contributes to ß-cell dysfunction and apoptosis through the activation of a terminal unfolded protein response (UPR). Our results uncover a new role for Bax Inhibitor-One (BI-1), a negative regulator of inositol-requiring enzyme 1 (IRE1α) in preserving ß-cell health against terminal UPR-induced apoptosis and pyroptosis in the context of supraphysiological loads of insulin production. BI-1-deficient mice experience a decline in endocrine pancreatic function in physiological and pathophysiological conditions, namely obesity induced by high-fat diet (HFD). We observed early-onset diabetes characterized by hyperglycemia, reduced serum insulin levels, ß-cell loss, increased pancreatic lipases and pro-inflammatory cytokines, and the progression of metabolic dysfunction. Pancreatic section analysis revealed that BI-1 deletion overburdens unfolded proinsulin in the ER of ß-cells, confirmed by ultrastructural signs of ER stress with overwhelmed IRE1α endoribonuclease (RNase) activity in freshly isolated islets. ER stress led to ß-cell dysfunction and islet loss, due to an increase in immature proinsulin granules and defects in insulin crystallization with the presence of Rod-like granules. These results correlated with the induction of autophagy, ER phagy, and crinophagy quality control mechanisms, likely to alleviate the atypical accumulation of misfolded proinsulin in the ER. In fine, BI-1 in ß-cells limited IRE1α RNase activity from triggering programmed ß-cell death through apoptosis and pyroptosis (caspase-1, IL-1ß) via NLRP3 inflammasome activation and metabolic dysfunction. Pharmaceutical IRE1α inhibition with STF-083010 reversed ß-cell failure and normalized the metabolic phenotype. These results uncover a new protective role for BI-1 in pancreatic ß-cell physiology as a stress integrator to modulate the UPR triggered by accumulating unfolded proinsulin in the ER, as well as autophagy and programmed cell death, with consequences on ß-cell function and insulin secretion. In pancreatic ß-cells, BI-1-/- deficiency perturbs proteostasis with proinsulin misfolding, ER stress, terminal UPR with overwhelmed IRE1α/XBP1s/CHOP activation, inflammation, ß-cell programmed cell death, and diabetes.
Subject(s)
Apoptosis , Endoplasmic Reticulum Stress , Insulin-Secreting Cells , Membrane Proteins , Proinsulin , Proteostasis , Unfolded Protein Response , Animals , Mice , Diet, High-Fat , Endoribonucleases/metabolism , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Membrane Proteins/metabolism , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Proinsulin/metabolism , Protein FoldingABSTRACT
Metastatic uveal melanomas are highly resistant to all existing treatments. To address this critical issue, we performed a kinome-wide CRISPR-Cas9 knockout screen, which revealed the LKB1-SIK2 module in restraining uveal melanoma tumorigenesis. Functionally, LKB1 loss enhances proliferation and survival through SIK2 inhibition and upregulation of the sodium/calcium (Na+ /Ca2+ ) exchanger SLC8A1. This signaling cascade promotes increased levels of intracellular calcium and mitochondrial reactive oxygen species, two hallmarks of cancer. We further demonstrate that combination of an SLC8A1 inhibitor and a mitochondria-targeted antioxidant promotes enhanced cell death efficacy in LKB1- and SIK2-negative uveal melanoma cells compared to control cells. Our study also identified an LKB1-loss gene signature for the survival prognostic of patients with uveal melanoma that may be also predictive of response to the therapy combination. Our data thus identify not only metabolic vulnerabilities but also new prognostic markers, thereby providing a therapeutic strategy for particular subtypes of metastatic uveal melanoma.
Subject(s)
Melanoma , Uveal Neoplasms , Humans , Calcium , Cell Proliferation , Melanoma/drug therapy , Reactive Oxygen Species , Uveal Neoplasms/genetics , Uveal Neoplasms/pathologyABSTRACT
Cholesterol efflux pathways could be exploited in tumor biology to unravel cancer vulnerabilities. A mouse model of lung-tumor-bearing KRASG12D mutation with specific disruption of cholesterol efflux pathways in epithelial progenitor cells promoted tumor growth. Defective cholesterol efflux in epithelial progenitor cells governed their transcriptional landscape to support their expansion and create a pro-tolerogenic tumor microenvironment (TME). Overexpression of the apolipoprotein A-I, to raise HDL levels, protected these mice from tumor development and dire pathologic consequences. Mechanistically, HDL blunted a positive feedback loop between growth factor signaling pathways and cholesterol efflux pathways that cancer cells hijack to expand. Cholesterol removal therapy with cyclodextrin reduced tumor burden in progressing tumor by suppressing the proliferation and expansion of epithelial progenitor cells of tumor origin. Local and systemic perturbations of cholesterol efflux pathways were confirmed in human lung adenocarcinoma (LUAD). Our results position cholesterol removal therapy as a putative metabolic target in lung cancer progenitor cells.
Subject(s)
Lung Neoplasms , Proto-Oncogene Proteins p21(ras) , Humans , Mice , Animals , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Cholesterol/metabolism , Lung Neoplasms/genetics , Cell Proliferation , Lung , Stem Cells/metabolism , Apolipoprotein A-I/metabolism , Tumor MicroenvironmentABSTRACT
Macrophages are pivotal in the initiation and development of atherosclerotic cardiovascular diseases. Recent studies have reinforced the importance of mitochondria in metabolic and signaling pathways to maintain macrophage effector functions. In this review, we discuss the past and emerging roles of macrophage mitochondria metabolic diversity in atherosclerosis and the potential avenue as biomarker. Beyond metabolic functions, mitochondria are also a signaling platform integrating epigenetic, redox, efferocytic and apoptotic regulations, which are exquisitely linked to their dynamics. Indeed, mitochondria functions depend on their density and shape perpetually controlled by mitochondria fusion/fission and biogenesis/mitophagy balances. Mitochondria can also communicate with other organelles such as the endoplasmic reticulum through mitochondria-associated membrane (MAM) or be secreted for paracrine actions. All these functions are perturbed in macrophages from mouse or human atherosclerotic plaques. A better understanding and integration of how these metabolic and signaling processes are integrated and dictate macrophage effector functions in atherosclerosis may ultimately help the development of novel therapeutic approaches.
Subject(s)
Atherosclerosis , Plaque, Atherosclerotic , Animals , Atherosclerosis/metabolism , Endoplasmic Reticulum , Humans , Macrophages/metabolism , Mice , Mitochondria , Plaque, Atherosclerotic/metabolismABSTRACT
RATIONALE: Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. OBJECTIVE: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. METHODS AND RESULTS: We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. CONCLUSIONS: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.
Subject(s)
Cholesterol/metabolism , Inflammation/metabolism , Lysosomes/metabolism , Macrophages/metabolism , Oxysterols/metabolism , Sterol Esterase/metabolism , Animals , Apoptosis , Biological Transport , Cholesterol Esters/metabolism , Erythrocytes/metabolism , Hydrolysis , Hypercholesterolemia/metabolism , Inflammasomes/metabolism , Liver X Receptors/metabolism , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Neuropeptides/metabolism , Receptors, LDL/metabolism , Splenomegaly/metabolism , Sterol Esterase/antagonists & inhibitors , rac1 GTP-Binding Protein/metabolismABSTRACT
This paper reports: (i) the facile synthesis of a cysteine synthon incorporating both a fluorescent group and a triphenylphosphonium derivative (TBTP) via the formation of a disulphide bond, which can subsequently undergo facile intracellular scission, (ii) the direct conjugation of this synthon to a non-permeable drug, (a cyclic PNA (peptide nucleic acid)-based compound has been chosen as a model), and (iii) that this conjugation enables the efficient homogenous delivery of the otherwise non-permeable cyclic PNA into the cytoplasm of cells, as demonstrated by fluorescence microscopy. Our results indicate that this fluorescent-labelled cysteine-TBTP synthon can provide a very useful tool for exploring the cellular uptake of a large range of molecules of biological interest, containing only a single reactive function. The preparation of an activated TBTP derivative is also described and this procedure could be widely used to introduce a TBTP cation to any thio-containing molecule.
Subject(s)
Drug Carriers/chemical synthesis , Drug Carriers/pharmacokinetics , Organophosphorus Compounds/pharmacokinetics , Peptide Nucleic Acids/administration & dosage , Cell Membrane Permeability , Cells , Cysteine/chemistry , Cytoplasm/metabolism , Fluorescent Dyes , HeLa Cells , Humans , Microscopy, Fluorescence , Organophosphorus Compounds/chemistryABSTRACT
A cyclic hexameric PNA-based compound labeled with fluorescein has been prepared following the liquid phase FPB strategy. Its cellular uptake, without and with electroporation, has been investigated by fluorescence microscopy.