Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity (original) (raw)
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A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB
The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB-/- cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stres...
F1000 - Post-publication peer review of the biomedical literature, 2015
The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca 2+ signaling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca 2+ release via mucolipin 1 (MCOLN1) activates calcineurin, which binds and de-phosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Molecular Pharmacology
Lysosomes degrade cellular proteins and organelles and regulate cell signaling by providing a surface for the formation of critical protein complexes, notably molecular target of rapamycin (mTOR) complex 1 (mTORC1). Striking differences in the lysosomes of cancer versus normal cells suggest that they could be targets for drug development. Although the lysomotropic drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been widely investigated, studies have focused on their ability to inhibit autophagy. We synthesized a novel compound, called EAD1, which is structurally related to CQ but is a 14-fold more potent inhibitor of cell proliferation. Here we find that EAD1 causes rapid relocation, membrane permeabilization (LMP), and deacidification of lysosomes, and it induces apoptosis and irreversibly blocks proliferation of human lung cancer H460, H520, H1299, HCC827, and H1703 cells. EAD1 causes dissociation of mTOR from lysosomes and increases mTOR's perinuclear versus cytoplasmic localization, changes previously shown to inactivate mTORC1. The effect on mTOR was not seen with HCQ, even at .10-fold greater concentrations. Phosphorylation of a downstream target of mTORC1, ribosomal protein S6, was inhibited by EAD1. Although EAD1 also inhibited autophagy, it retained full antiproliferative activity in autophagy-deficient H1650 lung cancer cells, which have a biallelic deletion of Atg7, and in H460 Atg7knockout cells. As Atg7 is critical for the canonical autophagy pathway, it is likely that inhibition of autophagy is not how EAD1 inhibits cell proliferation. Further studies are needed to determine the relationship of LMP to mTORC1 disruption and their relative contributions to drug-induced cell death. These studies support the lysosome as an underexplored target for new drug development.
Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis
Oncotarget, 2017
We have recently shown that hydrophobic weak base anticancer drugs are highly sequestered in acidic lysosomes, inducing TFEB-mediated lysosomal biogenesis and markedly increased lysosome numbers per cell. This enhanced lysosomal sequestration of chemotherapeutics, away from their intracellular targets, provoked cancer multidrug resistance. However, little is known regarding the fate of lysosome-sequestered drugs. While we suggested that sequestered drugs might be expelled from cancer cells via lysosomal exocytosis, no actual drug-induced lysosomal exocytosis was demonstrated. By following the subcellular localization of lysosomes during exposure to lysosomotropic chemotherapeutics, we herein demonstrate that lysosomal drug accumulation results in translocation of lysosomes from the perinuclear zone towards the plasma membrane via movement on microtubule tracks. Furthermore, following translocation to the plasma membrane in drug-treated cells, lysosomes fused with the plasma membrane and released their cargo to the extracellular milieu, as also evidenced by increased levels of the lysosomal enzyme cathepsin D in the extracellular milieu. These findings suggest that lysosomal exocytosis of chemotherapeutic drugloaded lysosomes is a crucial component of lysosome-mediated cancer multidrug resistance. We further argue that drug-induced lysosomal exocytosis bears important implications on tumor progression, as several lysosomal enzymes were found to play a key role in tumor cell invasion, angiogenesis and metastasis.
Activation of lysosomal mediated cell death in the course of autophagy by mTORC1 inhibitor
Scientific Reports, 2022
Lysosomal biogenesis plays a vital role in cell fate. Under certain conditions, excessive lysosomal biogenesis leads to susceptibility for lysosomal membrane permeabilization resulting in various pathological conditions including cell death. In cancer cells apoptosis machinery becomes dysregulated during the course of treatment, thus allows cancer cells to escape apoptosis. So it is therefore imperative to identify cytotoxic agents that exploit non-apoptotic mechanisms of cell death. Our study showed that pancreatic cancer cells treated with SDS-203 triggered an incomplete autophagic response and a nuclear translocation of transcriptional factor TFEB. This resulted in abundant biosynthesis and accumulation of autophagosomes and lysosomes into the cells leading to their death. It was observed that the silencing of autophagy genes didn’t alter the cell fate, whereas siRNA-mediated silencing of TFEB subdued SDS-203 mediated lysosomal biogenesis and associated cell death. Further mouse ...
Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB
Nature Cell Biology, 2015
The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca 2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca 2+ release through mucolipin 1 (MCOLN1) activates calcineurin, which binds and dephosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through TFEB required MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.
Lysosomal Regulation of mTORC1 by Amino Acids in Mammalian Cells
Biomolecules
The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth in eukaryotic cells. The active mTORC1 promotes cellular anabolic processes including protein, pyrimidine, and lipid biosynthesis, and inhibits catabolic processes such as autophagy. Consistent with its growth-promoting functions, hyper-activation of mTORC1 signaling is one of the important pathomechanisms underlying major human health problems including diabetes, neurodegenerative disorders, and cancer. The mTORC1 receives multiple upstream signals such as an abundance of amino acids and growth factors, thus it regulates a wide range of downstream events relevant to cell growth and proliferation control. The regulation of mTORC1 by amino acids is a fast-evolving field with its detailed mechanisms currently being revealed as the precise picture emerges. In this review, we summarize recent progress with respect to biochemical and biological findings in the regulation of mTORC1 signaling on the lysosomal membrane by amino acids.
Rapamycin directly activates lysosomal mucolipin TRP channels independent of mTOR
PLOS Biology
Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosomelocalized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca 2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis.
Journal of Biological Chemistry, 2013
Background: Lysosomes are required for autophagic degradation, which can be suppressed by lysosome inhibitors. Results: Inhibition of lysosome function resulted in autophagy activation via down-regulation of MTORC1. Conclusion: Lysosomes can affect autophagy initiation in addition to its role in autophagy degradation. Significance: The finding expands lysosome function to include regulation of autophagy activation and indicates a dual effect of lysosome inhibitors in autophagy. Autophagy can be activated via MTORC1 down-regulation by amino acid deprivation and by certain chemicals such as rapamycin, torin, and niclosamide. Lysosome is the degrading machine for autophagy but has also been linked to MTORC1 activation through the Rag/RRAG GTPase pathway. This association raises the question of whether lysosome can be involved in the initiation of autophagy. Toward this end, we found that niclosamide, an MTORC1 inhibitor, was able to inhibit lysosome degradation and increase lysosomal permeability. Niclosamide was ineffective in inhibiting MTORC1 in cells expressing constitutively activated Rag proteins, suggesting that its inhibitory effects were targeted to the Rag-MTORC1 signaling system. This places niclosamide in the same category of bafilomycin A 1 and concanamycin A, inhibitors of the vacuolar H ؉-ATPase, for its dependence on Rag GTPase in suppression of MTORC1. Surprisingly, classical lysosome inhibitors such as chloroquine, E64D, and pepstatin A were also able to inhibit MTORC1 in a Rag-dependent manner. These lysosome inhibitors were able to activate early autophagy events represented by ATG16L1 and ATG12 puncta formation. Our work established a link between the functional status of the lysosome in general to the Rag-MTORC1 signaling axis and autophagy activation. Thus, the lysosome is not only required for autophagic degradation but also affects autophagy activation. Lysosome inhibitors can have a dual effect in suppressing autophagy degradation and in initiating autophagy. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved mechanism by which cytoplasmic materials can be transported to and degraded in the lysosome. Autophagy serves to provide nutrients to starving cells or to remove superfluous subcellular organelles, aggregated proteins, or intracellular pathogens (1). As an important pathophysiological regulation mechanism during development, aging, and pathogenesis, autophagy can be induced or suppressed by a variety of signaling events. However, many of the signaling pathways have not been well defined. In general, autophagy can be induced by MTOR complex 1 (MTORC1) 2-dependent pathway or-independent pathway (2, 3). MTORC1 negatively regulates autophagic initiation, and many agents can thus induce autophagy by suppressing MTORC1 (2). A commonly used physiological autophagy stimulus is the deprivation of nutrients such as amino acids, which inactivates MTORC1. Rapamycin and Torin 1 are well defined small molecule chemicals that inhibit MTORC1 (4). Niclosamide is another chemical recently found to be able to induce autophagy and inhibit MTORC1 (5). The latter was attributed to the ability of niclosamide to cause cytoplasmic acidification by releasing protons from lysosomes (6). The lysosome has recently been found to play a uniquely important role in MTORC1 activation (7). Activation of MTORC1 by amino acids depends on the Rag/RRAG family of GTPase, which is found on the lysosomal membrane (8, 9). The Rag proteins are composed of RagA, RagB, RagC, and RagD.