The AXL-PYK2-PKCα axis as a nexus of stemness circuits in TNBC

Cancer stem cells (CSCs) are implicated in tumor initiation, metastasis and drug resistance, and considered as attractive targets for cancer therapy. Here we identified a clinically relevant signaling nexus mediated by AXL receptor, PYK2 and PKCα and show its impact on stemness in TNBC. AXL, PYK2, and PKCα expression correlates with stemness signature in basal-like breast cancer patients, and their depletion in multiple mesenchymal TNBC cell lines markedly reduced the number of mammosphere-forming cells and cells harboring CSCs characteristic markers. Knockdown of PYK2 reduced the levels of AXL, PKCα, FRA1, and PYK2 proteins, and similar trend was obtained upon PKCα depletion. PYK2 depletion decreased AXL transcription through feedback loops mediated by FRA1 and TAZ, whereas PKCα inhibition induced redistribution of AXL to endosomal/lysosomal compartment and enhanced its degradation. PYK2 and PKCα cooperate at a convergence point of multiple stemness-inducing pathways to regulate AXL levels and concomitantly the levels/activation of STAT3, TAZ, FRA1, and SMAD3 as well as the pluripotent transcription factors Nanog and Oct4. Induction of stemness in TNBC sensitized cells to PYK2 and PKCα inhibition suggesting that targeting the AXL-PYK2-PKCα circuit could be an efficient strategy to eliminate CSCs in TNBC.

Lysosome (Dys)function in Atherosclerosis—A Big Weight on the Shoulders of a Small Organelle

Atherosclerosis is a progressive insidious chronic disease that underlies most of the cardiovascular pathologies, including myocardial infarction and ischemic stroke. The malfunctioning of the lysosomal compartment has a central role in the etiology and pathogenesis of atherosclerosis. Lysosomes are the degradative organelles of mammalian cells and process endogenous and exogenous substrates in a very efficient manner. Dysfunction of these organelles and consequent inefficient degradation of modified low-density lipoproteins (LDL) and apoptotic cells in atherosclerotic lesions have, therefore, numerous deleterious consequences for cellular homeostasis and disease progression. Lysosome dysfunction has been mostly studied in the context of the inherited lysosomal storage disorders (LSDs). However, over the last years it has become increasingly evident that the consequences of this phenomenon are more far-reaching, also influencing the progression of multiple acquired human pathologies, such as neurodegenerative diseases, cancer, and cardiovascular diseases (CVDs). During the formation of atherosclerotic plaques, the lysosomal compartment of the various cells constituting the arterial wall is under severe stress, due to the tremendous amounts of lipoproteins being processed by these cells. The uncontrolled uptake of modified lipoproteins by arterial phagocytic cells, namely macrophages and vascular smooth muscle cells (VSMCs), is the initial step that triggers the pathogenic cascade culminating in the formation of atheroma. These cells become pathogenic “foam cells,” which are characterized by dysfunctional lipid-laden lysosomes. Here, we summarize the current knowledge regarding the origin and impact of the malfunctioning of the lysosomal compartment in plaque cells. We further analyze how the field of LSD research may contribute with some insights to the study of CVDs, particularly how therapeutic approaches that target the lysosomes in LSDs could be applied to hamper atherosclerosis progression and associated mortality.

Identification of the interferon-inducible GTPase GBP1 as major restriction factor for the Hepatitis E virus

This study aims to gain deeper insight into HEV-induced innate immunity by characterizing the crosstalk between the virus and the host factor guanylate-binding protein 1 (GBP1). We observe that the amount of GBP1 is elevated upon infection, although number of transcripts is decreased, which is explained by a prolonged protein half-life. Modulation of GBP1 levels via overexpression significantly inhibits the viral life cycle. Use of various GBP-1 mutants revealed that the antiviral effect of GBP-1 on HEV is independent from the GTPase-activity, but depends on the capacity of GBP-1 to form GBP1 homodimers. This connects GBP-1 to the autophagosomal pathway. Indeed, dimerization competent GBP1 targets the viral capsid protein to the lysosomal compartment leading to inactivation of the viral particle. Most importantly, silencing of GBP1 abolishes the antiviral effect of IFNγ on HEV. In IFNγ treated cells the virus is targeted to lysosomal structures and destroyed therein. This process depends in part on GBP1. These observations about the relevance of GBP1 for type II interferon-mediated innate immunity against HEV could be a base for tailoring novel antivirals and improvement of disease management. IMPORTANCE Although HEV represents a worldwide public health problem with 20 million infections and 44.000 death cases per year, there are still no specific antivirals available and many aspects of the viral life cycle are not well understood. Here we identify the guanylate binding protein 1 (GBP1) as a restriction factor affecting life cycle of HEV. Surprisingly, the antiviral effect of GBP1 does not depend on its GTPase function, but on its capacity to homodimerize. We revealed that GBP1 exerts its antiviral activity by targeting HEV to the lysosomal compartment where the virus is inactivated. Most importantly, we observed that the antiviral effect of interferon-γ on HEV strongly depends on GBP1. Our observation that GBP1 impairs HEV and is crucial for the antiviral effect of interferons on HEV extends understanding of host defense-mechanisms. As the interferon-system represents a universal defense-mechanism, our study could help to design novel antivirals targeting.

Fronto-temporal dementia risk gene TMEM106B has opposing effects in different lysosomal storage disorders

TMEM106B is a transmembrane protein localized to the endo-lysosomal compartment. Genome-wide association studies have identified TMEM106B as a risk modifier of Alzheimer’s disease and frontotemporal lobar degeneration, especially with progranulin haploinsufficiency. We recently demonstrated that TMEM106B loss rescues progranulin null mouse phenotypes including lysosomal enzyme dysregulation, neurodegeneration and behavioural alterations. However, the reason whether TMEM106B is involved in other neurodegenerative lysosomal diseases is unknown. Here, we evaluate the potential role of TMEM106B in modifying the progression of lysosomal storage disorders using progranulin-independent models of Gaucher disease and neuronal ceroid lipofuscinosis. To study Gaucher disease, we employ a pharmacological approach using the inhibitor conduritol B epoxide in wild-type and hypomorphic Tmem106b−/− mice. TMEM106B depletion ameliorates neuronal degeneration and some behavioural abnormalities in the pharmacological model of Gaucher disease, similar to its effect on certain progranulin null phenotypes. In order to examine the role of TMEM106B in neuronal ceroid lipofuscinosis, we crossbred Tmem106b−/− mice with Ppt1−/−, a genetic model of the disease. In contrast to its conduritol B epoxide-rescuing effect, TMEM106B loss exacerbates Purkinje cell degeneration and motor deficits in Ppt1−/− mice. Mechanistically, TMEM106B is known to interact with subunits of the vacuolar ATPase and influence lysosomal acidification. In the pharmacological Gaucher disease model, the acidified lysosomal compartment is enhanced and TMEM106B loss rescues in vivo phenotypes. In contrast, gene-edited neuronal loss of Ppt1 causes a reduction in vacuolar ATPase levels and impairment of the acidified lysosomal compartment, and TMEM106B deletion exacerbates the mouse Ppt1−/− phenotype. Our findings indicate that TMEM106B differentially modulates the progression of the lysosomal storage disorders Gaucher disease and neuronal ceroid lipofuscinosis. The effect of TMEM106B in neurodegeneration varies depending on vacuolar ATPase state and modulation of lysosomal pH. These data suggest TMEM106B as a target for correcting lysosomal pH alterations, and in particular for therapeutic intervention in Gaucher disease and neuronal ceroid lipofuscinosis.

TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling

Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and sequentially triggers two signaling cascades: the first one involving TIRAP and MyD88 adaptor proteins is induced in the plasma membrane, whereas the second engaging adaptor proteins TRAM and TRIF begins in early endosomes after endocytosis of the receptor. The LPS-induced internalization of TLR4 and hence also the activation of the TRIF-dependent pathway is governed by a GPI-anchored protein, CD14. The endocytosis of TLR4 terminates the MyD88-dependent signaling, while the following endosome maturation and lysosomal degradation of TLR4 determine the duration and magnitude of the TRIF-dependent one. Alternatively, TLR4 may return to the plasma membrane, which process is still poorly understood. Therefore, the course of the LPS-induced pro-inflammatory responses depends strictly on the rates of TLR4 endocytosis and trafficking through the endo-lysosomal compartment. Notably, prolonged activation of TLR4 is linked with several hereditary human diseases, neurodegeneration and also with autoimmune diseases and cancer. Recent studies have provided ample data on the role of diverse proteins regulating the functions of early, late, and recycling endosomes in the TLR4-induced inflammation caused by LPS or phagocytosis of E. coli. In this review, we focus on the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and also of LPS, in immune cells and discuss how dysregulation of the endo-lysosomal compartment contributes to the development of diverse human diseases.

Challenges in Tracking of Fluorochrome-Labelled Nanoparticles in Mice via Whole Body NIRF Imaging

Fluorochrome-labelled iron oxide magnetic nanoparticles (MNP) have been of great help in elucidating biological processes. Here, we used dually-fluorochrome-labelled MNP and studied to what extent fluorescence detection could reflect their fate in living animals. One day after application in mice (200 µmol Fe/kg body weight), the fluorescence of the dye attached to the core (DY-730) was very prominent and in agreement with the increase of iron in the liver and spleen of mice, but inconspicuous at time points thereafter. We attribute this fluorescence behavior to early degradation processes of the MNP´s core in the cellular lysosomal compartment. In contrast, the fluorescence of the dye DY-555 stuck to the PEG coating was not detectable in vivo. In summary, labelling of MNP with dyes at their metallic core could be of help when detecting first incidences of MNP biodegradation in vivo, as opposed to dyes attached to the MNP coating.

Rapamycin Re-Directs Lysosome Network, Stimulates ER-Remodeling, Involving Membrane CD317 and Affecting Exocytosis, in Campylobacter Jejuni-Lysate-Infected U937 Cells

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain–Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER–lysosome and ER–mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.

Lysosomal Fusion: An Efficient Mechanism Increasing Their Sequestration Capacity for Weak Base Drugs without Apparent Lysosomal Biogenesis

Lysosomal sequestration of anticancer therapeutics lowers their cytotoxic potential, reduces drug availability at target sites, and contributes to cancer resistance. Only recently has it been shown that lysosomal sequestration of weak base drugs induces lysosomal biogenesis mediated by activation of transcription factor EB (TFEB) which, in turn, enhances their accumulation capacity, thereby increasing resistance to these drugs. Here, we addressed the question of whether lysosomal biogenesis is the only mechanism that increases lysosomal sequestration capacity. We found that lysosomal sequestration of some tyrosine kinase inhibitors (TKIs), gefitinib (GF) and imatinib (IM), induced expansion of the lysosomal compartment. However, an expression analysis of lysosomal genes, including lysosome-associated membrane proteins 1, 2 (LAMP1, LAMP2), vacuolar ATPase subunit B2 (ATP6V1B2), acid phosphatase (ACP), and galactosidase beta (GLB) controlled by TFEB, did not reveal increased expression. Instead, we found that both studied TKIs, GF and IM, induced lysosomal fusion which was dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) mediated Ca2+signaling. A theoretical analysis revealed that lysosomal fusion is sufficient to explain the enlargement of lysosomal sequestration capacity. In conclusion, we demonstrated that extracellular TKIs, GF and IM, induced NAADP/Ca2+ mediated lysosomal fusion, leading to enlargement of the lysosomal compartment with significantly increased sequestration capacity for these drugs without apparent lysosomal biogenesis.

Bafilomycin A1 and U18666A Efficiently Impair ZIKV Infection

Zika virus (ZIKV) is a highly transmissive virus that belongs to the Flaviviridae family, which comprises several other pathogens that threaten human health. This re-emerging virus gained attention during the outbreak in Brazil in 2016, where a considerable number of microcephaly cases in newborns was associated with ZIKV infection during pregnancy. Lacking a preventive vaccine or antiviral drugs, efforts have been made to better understand the viral life cycle. In light of this, the relevance of the endosomal–lysosomal compartment for the ZIKV life cycle was investigated. A549 and SH-SY5Y cells were infected with either the African strain (associated with mild symptoms) or the French Polynesia strain (associated with neurological complications). For both strains, the V-ATPase inhibitor, bafilomycin A1, efficiently inhibited ZIKV entry and prevented the spread of the infection by interfering with viral maturation. Additionally, affecting cholesterol metabolism and transport with the drug U18666A, which inactivates late endosomes and lysosomes, impairs the viral life cycle. The data presented show a clear antiviral effect of two compounds that target the same compartments in different ways. This highlights the relevance of the endosomal–lysosomal compartment for the viral life cycle that should be considered as a target for antivirals.