Carnosine facilitates lysosomal release of inhibitors of T cell surveillance.

Cancer metabolism produces large fluxes of lactate and H+, which are extruded by membrane transporters. However, H+ production and extrusion must be coupled by diffusion, facilitated by mobile buffers. Yan et al. propose that carnosine, generated by CARNS2, provides this mobile buffering and enables lysosomal functions that block T cell surveillance.

Cationic amphiphilic drugs induce accumulation of cytolytic lysoglycerophospholipids in the lysosomes of cancer cells and block their recycling into common membrane glycerophospholipids.

Lysosomes are acidic organelles responsible for lipid catabolism, and their functions can be disrupted by cationic amphiphilic drugs that neutralize lumenal pH and thereby inhibit most lysosomal hydrolases. These drugs can also induce lysosomal membrane permeabilization and cancer cell death, but the underlying mechanism remains elusive. Here, we uncover that the cationic amphiphilic drugs induce a substantial accumulation of cytolytic lysoglycerophospholipids within the lysosomes of cancer cells, and thereby prevent the recycling of lysoglycerophospholipids to produce common membrane glycerophospholipids. Using quantitative mass spectrometry-based shotgun lipidomics, we demonstrate that structurally diverse cationic amphiphilic drugs, along with other types of lysosomal pH-neutralizing reagents, elevate the amounts of lysoglycerophospholipids in MCF7 breast carcinoma cells. Lysoglycerophospholipids constitute ∼11 mol% of total glycerophospholipids in lysosomes purified from MCF7 cells, compared with ∼1 mol% in the cell lysates. Treatment with cationic amphiphilic drug siramesine further elevates the lysosomal lysoglycerophospholipid content to ∼24 mol% of total glycerophospholipids. Exogenously added traceable lysophosphatidylcholine is rapidly acylated to form diacylphosphatidylcholine, but siramesine treatment sequesters the lysophosphatidylcholine in the lysosomes and prevents it from undergoing acylation. These findings shed light on the unexplored role of lysosomes in the recycling of lysoglycerophospholipids and uncover the mechanism of action of promising anticancer agents.

Activation of invasion by oncogenic reprogramming of cholesterol metabolism via increased NPC1 expression and macropinocytosis.

Cancer cells are dependent on cholesterol, and they possess strictly controlled cholesterol homeostasis mechanisms. These allow them to smoothly switch between cholesterol synthesis and uptake to fulfill their needs and to adapt environmental changes. Here we describe a mechanism of how cancer cells employ oncogenic growth factor signaling to promote uptake and utilization of extracellular cholesterol via Myeloid Zinc Finger 1 (MZF1)-mediated Niemann Pick C1 (NPC1) expression and upregulated macropinocytosis. Expression of p95ErbB2, highly oncogenic, standard-treatment resistant form of ErbB2 mobilizes lysosomes and activates EGFR, invasion and macropinocytosis. This is connected to a metabolic shift from cholesterol synthesis to uptake due to macropinocytosis-enabled flow of extracellular cholesterol. NPC1 increase facilitates extracellular cholesterol uptake and is necessary for the invasion of ErbB2 expressing breast cancer spheroids and ovarian cancer organoids, indicating a regulatory role for NPC1 in the process. The ability to obtain cholesterol as a byproduct of increased macropinocytosis allows cancer cells to direct the resources needed for the energy-consuming cholesterol synthesis towards other activities such as invasion. These results demonstrate that macropinocytosis is not only an alternative energy source for cancer cells but also an efficient way to provide building material, such as cholesterol, for its macromolecules and membranes.

Increased mortality in patients with hematologic malignancies treated with proton pump inhibitors: a nationwide cohort study.

Proton Pump inhibitors (PPIs) are frequently prescribed to cancer patients to prevent gastric mucosal damage. Post-diagnostic PPI use in patients with solid tumors may be associated with increased cancer mortality. However, the hazardous impact of PPIs in patients with hematologic malignancies remains unknown. This association was investigated in a large, retrospective cohort study using data from the Danish nationwide health registries. The outcomes were cancer-specific or non-cancer deaths. We identified 15,320 patients with hematologic malignancies and of these 1811 were identified as post-diagnostic PPI users. PPI users had significantly increased HRs for cancer-specific mortality (HR 1.31; 95% CI, 1.18-1.44) and 1-year cancer-specific mortality (HR 1.50, 95% CI 1.29-1.74) as compared to nonusers. The association between PPI use and increased cancer-specific mortality in Danish patients with hematologic malignancies supports the raised concerns regarding the frequent use of PPIs in cancer patients.

Necrocide 1 mediates necrotic cell death and immunogenic response in human cancer cells.

Many anticancer agents induce apoptosis, mitotic catastrophe or cellular senescence. Here, we report the functional characterization of an experimental inducer of tumor necrosis factor (TNF)-independent necrosis, necrocide-1 (NC1). NC1 (but not its stereoisomer) killed a panel of human cancer cells (but not normal cells) at nanomolar concentrations and with a non-apoptotic, necrotic morphotype, both in vitro and in vivo. NC1-induced killing was not inhibited by caspase blockers, anti-apoptotic BCL2 overexpression or TNFα neutralization, suggesting that NC1 elicits a bona fide necrotic pathway. However, pharmacological or genetic inhibition of necroptosis, pyroptosis and ferroptosis failed to block NC1-mediated cell death. Instead, NC1 elicited reactive oxygen species (ROS) production by mitochondria, and elimination of mitochondrial DNA, quenching of mitochondrial ROS, as well as blockade of mitochondrial permeability transition with cyclosporine A, interfered with NC1-induced cell death. NC1 induced hallmarks of immunogenic cell death incurring calreticulin (CALR) exposure, ATP secretion and high mobility group box 1 (HMGB1) release. Taken together, these data identify a previously uncharacterized signaling cascade leading to an immunogenic variant of mitochondrion-regulated necrosis, supporting the notion that eliciting regulated necrosis may constitute a valid approach for anticancer therapy.

TFEB-dependent lysosome biogenesis is required for senescence.

The accumulation of senescent cells is recognised as a driver of tissue and organismal ageing. One of the gold-standard hallmarks of a senescent cell is an increase in lysosomal content, as measured by senescence-associated ß-galactosidase (Senß-Gal) activity. The lysosome plays a central role in integrating mitogenic and stress cues to control cell metabolism, which is known to be dysregulated in senescence. Despite this, little is known about the cause and consequence of lysosomal biogenesis in senescence. We find here that lysosomes in senescent cells are dysfunctional; they have higher pH, increased evidence of membrane damage and reduced proteolytic capacity. The significant increase in lysosomal content is however sufficient to maintain degradative capacity of the cell to a level comparable to proliferating control cells. We demonstrate that increased nuclear TFEB/TFE3 supports lysosome biogenesis, is a hallmark of multiple forms of senescence and is required for senescent cell survival. TFEB/TFE3 are hypo-phosphorylated and show constitutive nuclear localisation in senescence. Evidence suggests that several pathways may contribute to TFEB/TFE3 dysregulation in senescence.

Cationic amphiphilic antihistamines inhibit STAT3 via Ca2+-dependent lysosomal H+ efflux.

Commonly used antihistamines and other cationic amphiphilic drugs (CADs) are emerging as putative cancer drugs. Their unique chemical structure enables CADs to accumulate rapidly inside lysosomes, where they increase lysosomal pH, alter lysosomal lipid metabolism, and eventually cause lysosomal membrane permeabilization. Here, we show that CAD-induced rapid elevation in lysosomal pH is caused by a lysosomal H+ efflux that requires P2RX4-mediated lysosomal Ca2+ release and precedes the lysosomal membrane permeabilization. The subsequent cytosolic acidification triggers the dephosphorylation, lysosomal translocation, and inactivation of the oncogenic signal transducer and activator of transcription 3 (STAT3) transcription factor. Moreover, CAD-induced lysosomal H+ efflux sensitizes cancer cells to apoptosis induced by STAT3 inhibition and acts synergistically with STAT3 inhibition in restricting the tumor growth of A549 non-small cell lung carcinoma xenografts. These findings identify lysosomal H+ efflux and STAT3 inhibition as anticancer mechanisms of CADs and reinforce the repurposing of safe and inexpensive CADs as cancer drugs with a drug combination strategy.

Targeting Cancer Lysosomes with Good Old Cationic Amphiphilic Drugs.

Being originally discovered as cellular recycling bins, lysosomes are today recognized as versatile signaling organelles that control a wide range of cellular functions that are essential not only for the well-being of normal cells but also for malignant transformation and cancer progression. In addition to their core functions in waste disposal and recycling of macromolecules and energy, lysosomes serve as an indispensable support system for malignant phenotype by promoting cell growth, cytoprotective autophagy, drug resistance, pH homeostasis, invasion, metastasis, and genomic integrity. On the other hand, malignant transformation reduces the stability of lysosomal membranes rendering cancer cells sensitive to lysosome-dependent cell death. Notably, many clinically approved cationic amphiphilic drugs widely used for the treatment of other diseases accumulate in lysosomes, interfere with their cancer-promoting and cancer-supporting functions and destabilize their membranes thereby opening intriguing possibilities for cancer therapy. Here, we review the emerging evidence that supports the supplementation of current cancer therapies with lysosome-targeting cationic amphiphilic drugs.

Annexin A7 mediates lysosome repair independently of ESCRT-III.

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair.

Ursolic Acid Impairs Cellular Lipid Homeostasis and Lysosomal Membrane Integrity in Breast Carcinoma Cells.

Cancer is one of the leading causes of death worldwide, thus the search for new cancer therapies is of utmost importance. Ursolic acid is a naturally occurring pentacyclic triterpene with a wide range of pharmacological activities including anti-inflammatory and anti-neoplastic effects. The latter has been assigned to its ability to promote apoptosis and inhibit cancer cell proliferation by poorly defined mechanisms. In this report, we identify lysosomes as the essential targets of the anti-cancer activity of ursolic acid. The treatment of MCF7 breast cancer cells with ursolic acid elevates lysosomal pH, alters the cellular lipid profile, and causes lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol. Lysosomal membrane permeabilization precedes the essential hallmarks of apoptosis placing it as an initial event in the cascade of effects induced by ursolic acid. The disruption of the lysosomal function impairs the autophagic pathway and likely partakes in the mechanism by which ursolic acid kills cancer cells. Furthermore, we find that combining treatment with ursolic acid and cationic amphiphilic drugs can significantly enhance the degree of lysosomal membrane permeabilization and cell death in breast cancer cells.

Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair.

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here, we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by the formation of contacts with the endoplasmic reticulum (ER), which depends on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP-ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to the failure of lysosomal membrane repair. The cholesterol-PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

Galactosyl- and glucosylsphingosine induce lysosomal membrane permeabilization and cell death in cancer cells.

Isomeric lysosphingolipids, galactosylsphingosine (GalSph) and glucosylsphingosine (GlcSph), are present in only minute levels in healthy cells. Due to defects in their lysosomal hydrolysis, they accumulate at high levels and cause cytotoxicity in patients with Krabbe and Gaucher diseases, respectively. Here, we show that GalSph and GlcSph induce lysosomal membrane permeabilization, a hallmark of lysosome-dependent cell death, in human breast cancer cells (MCF7) and primary fibroblasts. Supporting lysosomal leakage as a causative event in lysosphingolipid-induced cytotoxicity, treatment of MCF7 cells with lysosome-stabilizing cholesterol prevented GalSph- and GlcSph-induced cell death almost completely. In line with this, fibroblasts from a patient with Niemann-Pick type C disease, which is caused by defective lysosomal cholesterol efflux, were significantly less sensitive to lysosphingolipid-induced lysosomal leakage and cell death. Prompted by the data showing that MCF7 cells with acquired resistance to lysosome-destabilizing cationic amphiphilic drugs (CADs) were partially resistant to the cell death induced by GalSph and GlcSph, we compared these cell death pathways with each other. Like CADs, GalSph and GlcSph activated the cyclic AMP (cAMP) signalling pathway, and cAMP-inducing forskolin sensitized cells to cell death induced by low concentrations of lysosphingolipids. Contrary to CADs, lysosphingolipid-induced cell death was independent of lysosomal Ca2+ efflux through P2X purinerigic receptor 4. These data reveal GalSph and GlcSph as lysosome-destabilizing lipids, whose putative use in cancer therapy should be further investigated. Furthermore, the data supports the development of lysosome stabilizing drugs for the treatment of Krabbe and Gaucher diseases and possibly other sphingolipidoses.

Unraveling membrane properties at the organelle-level with LipidDyn.

Cellular membranes are formed from different lipids in various amounts and proportions depending on the subcellular localization. The lipid composition of membranes is sensitive to changes in the cellular environment, and its alterations are linked to several diseases. Lipids not only form lipid-lipid interactions but also interact with other biomolecules, including proteins. Molecular dynamics (MD) simulations are a powerful tool to study the properties of cellular membranes and membrane-protein interactions on different timescales and resolutions. Over the last few years, software and hardware for biomolecular simulations have been optimized to routinely run long simulations of large and complex biological systems. On the other hand, high-throughput techniques based on lipidomics provide accurate estimates of the composition of cellular membranes at the level of subcellular compartments. Lipidomic data can be analyzed to design biologically relevant models of membranes for MD simulations. Similar applications easily result in a massive amount of simulation data where the bottleneck becomes the analysis of the data. In this context, we developed LipidDyn, a Python-based pipeline to streamline the analyses of MD simulations of membranes of different compositions. Once the simulations are collected, LipidDyn provides average properties and time series for several membrane properties such as area per lipid, thickness, order parameters, diffusion motions, lipid density, and lipid enrichment/depletion. The calculations exploit parallelization, and the pipeline includes graphical outputs in a publication-ready form. We applied LipidDyn to different case studies to illustrate its potential, including membranes from cellular compartments and transmembrane protein domains. LipidDyn is available free of charge under the GNU General Public License from https://github.com/ELELAB/LipidDyn.

Lysosomal Changes in Mitosis.

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Systematical analysis reveals a strong cancer relevance of CREB1-regulated genes.

The transcription factor cyclic-AMP response element-binding protein 1 (CREB1) responds to cAMP level and controls the expression of target genes, which regulates nutrition partitioning. The promoters of CREB1-targeted genes responsive to cAMP have been extensively investigated and characterized with the presence of both cAMP response element and TATA box. Compelling evidence demonstrates that CREB1 also plays an essential role in promoting tumor development. However, only very few genes required for cell survival, proliferation and migration are known to be constitutively regulated by CREB1 in tumors. Their promoters mostly do not harbor any cAMP response element. Thus, it is very likely that CREB1 regulates the expressions of distinct sets of target genes in normal tissues and tumors. The whole gene network constitutively regulated by CREB1 in tumors has remained unrevealed. Here, we employ a systematical and integrative approach to decipher this gene network in the context of both tissue cultured cancer cells and patient samples. We combine transcriptomic, Rank-Rank Hypergeometric Overlap, and Chipseq analysis, to define and characterize CREB1-regulated genes in a multidimensional fashion. A strong cancer relevance of those top-ranked targets, which meet the most stringent criteria, is eventually verified by overall survival analysis of cancer patients. These findings strongly suggest the importance of genes constitutively regulated by CREB1 for their implicative involvement in promoting tumorigenesis.

They Might Cut It-Lysosomes and Autophagy in Mitotic Progression.

The division of one cell into two looks so easy, as if it happens without any control at all. Mitosis, the hallmark of mammalian life is, however, tightly regulated from the early onset to the very last phase. Despite the tight control, errors in mitotic division occur frequently and they may result in various chromosomal instabilities and malignancies. The flow of events during mitotic progression where the chromosomes condensate and rearrange with the help of the cytoskeletal network has been described in great detail. Plasma membrane dynamics and endocytic vesicle movement upon deadhesion and reattachment of dividing cells are also demonstrated to be functionally important for the mitotic integrity. Other cytoplasmic organelles, such as autophagosomes and lysosomes, have until recently been considered merely as passive bystanders in this process. Accordingly, at the onset of nuclear envelope breakdown in prometaphase, the number of autophagic structures and lysosomes is reduced and the bulk autophagic machinery is suppressed for the duration of mitosis. This is believed to ensure that the exposed nuclear components are not unintentionally delivered to autophagic degradation. With the evolving technologies that allow the detection of subtle alterations in cytoplasmic organelles, our understanding of the small-scale regulation of intracellular organelles has deepened rapidly and we discuss here recent discoveries revealing unexpected roles for autophagy and lysosomes in the preservation of genomic integrity during mitosis.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Identification of lysosome-targeting drugs with anti-inflammatory activity as potential invasion inhibitors of treatment resistant HER2 positive cancers.

PURPOSE: Most HER2 positive invasive cancers are either intrinsic non-responsive or develop resistance when treated with 1st line HER2 targeting drugs. Both 1st and 2nd line treatments of HER2 positive cancers are aimed at targeting the HER2 receptor directly, thereby strongly limiting the treatment options of HER2/ErbB2 inhibition resistant invasive cancers. METHODS: We used phenotypic high throughput microscopy screening to identify efficient inhibitors of ErbB2-induced invasion using 1st line HER2 inhibitor trastuzumab- and pertuzumab-resistant, p95-ErbB2 expressing breast cancer cells in conjunction with the Prestwick Chemical Library®. The screening entailed a drug's ability to inhibit ErbB2-induced, invasion-promoting positioning of lysosomes at the cellular periphery, a phenotype that defines their invasiveness. In addition, we used high throughput microscopy and biochemical assays to assess the effects of the drugs on lysosomal membrane permeabilization (LMP) and autophagy, two features connected to cancer treatment. Using 2nd line HER2 inhibitor lapatinib resistant 3-dimensional model systems, we assessed the effects of the drugs on ErbB2 positive breast cancer spheroids and developed a high-throughput invasion assay for HER2 positive ovarian cancer organoids for further evaluation. RESULTS: We identified Auranofin, Colchicine, Monensin, Niclosamide, Podophyllotoxin, Quinacrine and Thiostrepton as efficient inhibitors of invasive growth of 2nd line HER2 inhibitor lapatinib resistant breast cancer spheroids and ovarian cancer organoids. We classified these drugs into four groups based on their ability to target lysosomes by inducing autophagy and/or LMP, i.e., drugs inducing early LMP, early autophagy with late LMP, late LMP, or neither. CONCLUSIONS: Our results indicate that targetable lysosome-engaging cellular pathways downstream of ErbB2 contribute to invasion. They support lysosomal trafficking as an attractive target for therapy aiming at preventing the spreading of cancer cells. Since these drugs additionally possess anti-inflammatory activities, they could serve as multipurpose drugs simultaneously targeting infection/inflammation and cancer spreading.

Control of mitosis, inflammation, and cell motility by limited leakage of lysosomes.

Lysosomal membrane permeabilization and subsequent leakage of lysosomal hydrolases into the cytosol are considered as the major hallmarks of evolutionarily conserved lysosome-dependent cell death. Contradicting this postulate, new sensitive methods that can detect a minimal lysosomal membrane damage have demonstrated that lysosomal leakage does not necessarily equal cell death. Notably, cells are not only able to survive minor lysosomal membrane permeabilization, but some of their normal functions actually depend on leaked lysosomal hydrolases. Here we discuss emerging data suggesting that spatially and temporally controlled lysosomal leakage delivers lysosomal hydrolases to specific subcellular sites of action and controls at least three essential cellular processes, namely mitotic chromosome segregation, inflammatory signaling, and cellular motility.