Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity

Benny Zhitomirsky,Anna Yunaev,Yehuda G Assaraf,Michal Stark,Ariel Kaplan,Roman Kreiserman

doi:10.1038/s41419-018-1227-0

Benny Zhitomirsky, Anna Yunaev + Show 4 more

Open Access

https://doi.org/10.1038/s41419-018-1227-0

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Abstract

Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca2+ release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca2+, or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.

Highlights

Small molecules with hydrophobic weak base properties markedly accumulate in lysosomes via a mechanism known as ion trapping; due to their hydrophobic nature, Official journal of the Cell Death Differentiation AssociationZhitomirsky et al Cell Death and Disease (2018)9:1191 extrusion of the sequestered drugs from the cells[2,5,6,7].lysosomal sequestration of anticancer drugs triggered lysosomal biogenesis via activation and nuclear translocation of transcription factor EB (TFEB)[5], the master regulator of lysosomal biogenesis, and an activator of autophagy and lysosomal exocytosis[8,9,10,11]
We have recently demonstrated that lysosomal sequestration of anticancer drugs contributes to cancer drug resistance by reducing the concentration of these drugs at their cellular target sites and by activating lysosomal exocytosis, resulting in the Official journal of the Cell Death Differentiation Association
Based on these collective findings, we have proposed a novel model for drug-induced lysosome-mediated acquired drug resistance, in which lysosomal accumulation of anticancer drugs induces TFEB-mediated lysosomal biogenesis, an elevation in lysosome number per cell, and consequent chemoresistance due to increased lysosomal drug sequestration[5]

Summary

Introduction

Small molecules with hydrophobic weak base properties markedly accumulate in lysosomes via a mechanism known as ion trapping; due to their hydrophobic nature, Official journal of the Cell Death Differentiation AssociationZhitomirsky et al Cell Death and Disease (2018)9:1191 extrusion of the sequestered drugs from the cells[2,5,6,7].lysosomal sequestration of anticancer drugs triggered lysosomal biogenesis via activation and nuclear translocation of transcription factor EB (TFEB)[5], the master regulator of lysosomal biogenesis, and an activator of autophagy and lysosomal exocytosis[8,9,10,11]. We have shown that drug naïve cells with intrinsically higher lysosome number per cell, sequester lysosomotropic chemotherapeutics more efficiently away from their target sites when compared to cells with low-lysosome number per cell, which contributes to an enhanced intrinsic resistance to lysosomotropic chemotherapeutics. Based on these collective findings, we have proposed a novel model for drug-induced lysosome-mediated acquired drug resistance, in which lysosomal accumulation of anticancer drugs induces TFEB-mediated lysosomal biogenesis, an elevation in lysosome number per cell, and consequent chemoresistance due to increased lysosomal drug sequestration[5]. The activity of TFEB is inhibited via its phosphorylation on Ser[211] by mammalian target of rapamycin complex 1 (mTORC1)[13]

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