Lysosomal Gene Expression Research Articles

TSC1-mutant bladder cancer expression signature in relation to nuclear localization of TFE3 and potential for targetable dependency.

e16532 Background: Mutation and inactivation of the tumor suppressor gene TSC1 is a recurrent (6-10%) event in bladder cancer, but whether it functions as a driver event for tumor development has been uncertain. Methods: We performed differential gene expression and pathway analyses using RNA-seq data from the curated TCGA TSC1 mutant BLCA (n = 26) and TSC1 wild-type BLCA (n = 382) cohort and compared to an internal cohort of putative TSC1/TSC2-driven tumors (n = 63). Mechanistic studies, as well as RNA-seq and H3K27ac ChIP-seq analyses, were conducted in 2 TSC1 mutant/WT BLCA cell lines. Results: Comparison of The Cancer Genome Atlas (TCGA) TSC1-mutant bladder cancers ( TSC1mBLCA) with TCGA TSC1 wildtype tumors ( TSC1WTBLCA) identified a conserved TSC-associated expression signature, similar to ones seen in syndromic TSC tumors. GSEA and DESeq2 analyses implicated both mTORC1 hyperactivation, as well as activation of lysosomal pathways in TSC1mBLCA. We validated our findings by IHC analysis of a separate cohort of TSC1mBLCA (n = 5), compared to TSC1WTBLCA (n = 5). In addition, we found that TFE3, a transcriptional regulator of lysosomal gene expression, was relatively highly expressed in BLCA (compared to other MiT-TFE genes) and was localized to the nucleus in TSC1mBLCA but not in TSC1WTBLCA. Mechanistic studies of two TSC1mBLCA cell lines and their respective TSC1 addback derivatives, recapitulated the phenotype found in human tumors and demonstrated that TFE3 was both post-translationally modified and predominantly nuclear in TSC1-null cell lines compared to TSC1 addbacks. RNA-seq and H3K27ac ChIP-Seq analyses showed that TSC1mBLCA cell lines retained elements of the TSC-associated expression signature that was seen in TSC1mBLCA tumors, confirming differential activation of TFE3 in response to TSC1 loss. Nuclear localization of TFE3 in TSC1mBLCA cell lines was only partially reversed by rapamycin treatment and was unaffected by treatment of Torin1. SiRNA mediated knockdown of TFE3 significantly decreased cell growth and viability in TSC1mBLCA cell lines and did not result in compensatory upregulation of TFEB and MITF. Conclusions: Our findings indicate that TSC1 mutant bladder tumors retain elements of a conserved transcriptional signature that is characterized by nuclear localization and activation of TFE3. Aberrant TFE3 activation likely contributes to TSC1mBLCA development and may therefore be amenable to targeted therapy.

Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence.

Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and dependence on Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drive multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia toward distinct remodeling states and through Axl, alter microglial dependence on survival pathway, CSF1R.

Correlation of age of onset and clinical severity in Niemann–Pick disease type C1 with lysosomal abnormalities and gene expression

Niemann–Pick disease type C1 (NPC1) is a rare, prematurely fatal lysosomal storage disorder which exhibits highly variable severity and disease progression as well as a wide-ranging age of onset, from perinatal stages to adulthood. This heterogeneity has made it difficult to obtain prompt diagnosis and to predict disease course. In addition, small NPC1 patient sample sizes have been a limiting factor in acquiring genome-wide transcriptome data. In this study, primary fibroblasts from an extensive cohort of 41 NPC1 patients were used to validate our previous findings that the lysosomal quantitative probe LysoTracker can be used as a predictor for age of onset and disease severity. We also examined the correlation between these clinical parameters and RNA expression data from primary fibroblasts and identified a set of genes that were significantly associated with lysosomal defects or age of onset, in particular neurological symptom onset. Hierarchical clustering showed that these genes exhibited distinct expression patterns among patient subgroups. This study is the first to collect transcriptomic data on such a large scale in correlation with clinical and cellular phenotypes, providing a rich genomic resource to address NPC1 clinical heterogeneity and discover potential biomarkers, disease modifiers, or therapeutic targets.

Inositol triphosphate signaling triggers lysosome biogenesis via calcium release from endoplasmic reticulum stores.

Lysosomes serve as cellular degradation and signaling centers that coordinate the turnover of macromolecules with cell metabolism. The adaptation of cellular lysosome content and activity via the induction of lysosome biogenesis is therefore key to cell physiology and to counteract disease. Previous work has established a pathway for the induction of lysosome biogenesis in signaling-inactive starved cells that is based on the repression of mTORC1-mediated nutrient signaling. How lysosomal biogenesis is facilitated in signaling-active fed cells is poorly understood. A recent study by Malek et al (Malek et al, 2022) partially fills this gap by unraveling a nutrient signaling-independent pathway for lysosome biogenesis that operates in signaling-active cells. This pathway involves the receptor-mediated activation of phospholipase C, inositol (1,4,5)-triphosphate (IP3)-triggered release of calcium ions from endoplasmic reticulum stores, and the calcineurin-induced activation of transcription factor EB (TFEB) and its relative TFE3 to induce lysosomal gene expression independent of calcium in the lysosome lumen. These findings contribute to our understanding of how lysosome biogenesis and function are controlled in response to environmental changes and cell signaling and may conceivably be of relevance for our understanding and the treatment of lysosome-related diseases as well as for aging and neurodegeneration.

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

Subendothelial Accumulation of Exosomes and Coronary Microvascular Dysfunction in Mice Lacking Acid Ceramidase

Coronary microvascular dysfunction (CMD) has been demonstrated to be one of the major factors contributing to ischemic heart disease. However, the mechanisms initiating CMD remain poorly understood. The present study tested the hypothesis that lysosomal acid ceramidase finely controls subendothelial accumulation of exosomes leading CMD and subsequent myocardial ischemia. Using an endothelial-specific acid ceramidase (AC) gene (mouse gene code: Asah1) knockout (Asah1fl/fl/ECcre) mouse model fed with the Western diet (WD), we found that Asah1fl/fl/ECcre mice on the WD have more thickening of mid-layer in their small coronary arteries as shown significantly increased media/lumen ratio compared to the WD-fed wild type littermates (WT/WT). By nanoparticle tracking analysis (NTA) using NanoSight NS300 nanoparticle analyzer, we also observed that exosomes (~80-130 nm) in the blood stream were markedly increased in WD-fed Asah1fl/fl/ECcre mice compared to WT/WT mice. There were also more exosomes detected in the subendothelial space of coronary arterial wall, as shown by remarkable increase in colocalization of exosome marker CD63 or Annexin II with proteoglycans, bioglycan or decorin (subendothelial matrix markers) in Asah1fl/fl/ECcre mice. By electron microscopy (EM), we further confirmed that Asah1fl/fl/ECcre mice on the WD also significantly augmented the accumulation of exosomes under the endothelium small coronary arteries. Functionally, electrocardiogram (ECG) recording showed that in response to ergonovine, a coronary artery constrictor, the elevation of S-T segment was more remarkable in WD-fed Asah1fl/fl/ECcre than WT/WT mice. Using HypoxyProbe-1, the myocardial ischemic response to ergonovine was also detected more in the heart of WD-fed Asah1fl/fl/ECcre than WT/WT mice. Together, these results indicate that subendothelial exosome accumulation due to defect of lysosomal AC gene expression and activity may be a crucial pathogenic factor for CMD.

YAP plays a crucial role in the development of cardiomyopathy in lysosomal storage diseases.

Lysosomal dysfunction caused by mutations in lysosomal genes results in lysosomal storage disorder (LSD), characterized by accumulation of damaged proteins and organelles in cells and functional abnormalities in major organs, including the heart, skeletal muscle, and liver. In LSD, autophagy is inhibited at the lysosomal degradation step and accumulation of autophagosomes is observed. Enlargement of the left ventricle (LV) and contractile dysfunction were observed in RagA/B cardiac-specific KO (cKO) mice, a mouse model of LSD in which lysosomal acidification is impaired irreversibly. YAP, a downstream effector of the Hippo pathway, was accumulated in RagA/B cKO mouse hearts. Inhibition of YAP ameliorated cardiac hypertrophy and contractile dysfunction and attenuated accumulation of autophagosomes without affecting lysosomal function, suggesting that YAP plays an important role in mediating cardiomyopathy in RagA/B cKO mice. Cardiomyopathy was also alleviated by downregulation of Atg7, an intervention to inhibit autophagy, whereas it was exacerbated by stimulation of autophagy. YAP physically interacted with transcription factor EB (TFEB), a master transcription factor that controls autophagic and lysosomal gene expression, thereby facilitating accumulation of autophagosomes without degradation. These results indicate that accumulation of YAP in the presence of LSD promotes cardiomyopathy by stimulating accumulation of autophagosomes through activation of TFEB.

Age-related injury responses of human oligodendrocytes to metabolic insults: link to BCL-2 and autophagy pathways

Myelin destruction and oligodendrocyte (OL) death consequent to metabolic stress is a feature of CNS disorders across the age spectrum. Using cells derived from surgically resected tissue, we demonstrate that young (<age 5) pediatric-aged sample OLs are more resistant to in-vitro metabolic injury than fetal O4+ progenitor cells, but more susceptible to cell death and apoptosis than adult-derived OLs. Pediatric but not adult OLs show measurable levels of TUNEL+ cells, a feature of the fetal cell response. The ratio of anti- vs pro-apoptotic BCL-2 family genes are increased in adult vs pediatric (<age 5) mature OLs and in more mature OL lineage cells. Lysosomal gene expression was increased in adult and pediatric compared to fetal OL lineage cells. Cell death of OLs was increased by inhibiting pro-apoptotic BCL-2 gene and autophagy activity. These distinct age-related injury responses should be considered in designing therapies aimed at reducing myelin injury.

Induced Pluripotent Stem Cells to Understand Mucopolysaccharidosis. I: Demonstration of a Migration Defect in Neural Precursors

Background: Mucopolysaccharidosis type I-Hurler (MPS1-H) is a severe genetic lysosomal storage disorder due to loss-of-function mutations in the IDUA gene. The subsequent complete deficiency of alpha l-iduronidase enzyme is directly responsible of a progressive accumulation of glycosaminoglycans (GAG) in lysosomes which affects the functions of many tissues. Consequently, MPS1 is characterized by systemic symptoms (multiorgan dysfunction) including respiratory and cardiac dysfunctions, skeletal abnormalities and early fatal neurodegeneration. Methods: To understand mechanisms underlying MPS1 neuropathology, we generated induced pluripotent stem cells (iPSC) from a MPS1-H patient with loss-of-function mutations in both IDUA alleles. To avoid variability due to different genetic background of iPSC, we established an isogenic control iPSC line by rescuing IDUA expression by a lentivectoral approach. Results: Marked differences between MPS1-H and IDUA-corrected isogenic controls were observed upon neural differentiation. A scratch assay revealed a strong migration defect of MPS1-H cells. Also, there was a massive impact of IDUA deficiency on gene expression (340 genes with an FDR < 0.05). Conclusions: Our results demonstrate a hitherto unknown connection between lysosomal degradation, gene expression and neural motility, which might account at least in part for the phenotype of MPS1-H patients.

Genetics of Gene Expression in the Aging Human Brain Reveal TDP-43 Proteinopathy Pathophysiology

Here, we perform a genome-wide screen for variants that regulate the expression of gene co-expression modules in the aging human brain; we discover and replicate such variants in the TMEM106B and RBFOX1 loci. The TMEM106B haplotype is known to influence the accumulation of TAR DNA-binding protein 43kDa (TDP-43) proteinopathy, and the haplotype's large-scale transcriptomic effects include the dysregulation of lysosomal genes and alterations in synaptic gene splicing that are also seen in the pathophysiology of TDP-43 proteinopathy. Further, a variant near GRN, another TDP-43 proteinopathy susceptibility gene, shows concordant effects with the TMEM106B haplotype. Leveraging neuropathology data from the same participants, we also show that TMEM106B and APOE-amyloid-β effects converge to alter myelination and lysosomal gene expression, which then contributes to TDP-43 accumulation. These results advance our mechanistic understanding of the TMEM106B TDP-43 risk haplotype and uncover a transcriptional program that mediates the converging effects of APOE-amyloid-β and TMEM106B on TDP-43 aggregation in older adults.

Remote ischemic postconditioning attenuates damage in rats with chronic cerebral ischemia by upregulating the autophagolysosome pathway via the activation of TFEB

The transcription factor EB (TFEB) is known for its role in lysosomal biogenesis, and it coordinates this process by driving autophagy and lysosomal gene expression during ischemia. In the present study, we aimed to explore the role of the TFEB-regulated autophagolysosome pathway (ALP) in rats with chronic cerebral ischemia (CCI) that were treated with remote ischemic postconditioning (RIPC). A modified 2-vessel occlusion (2-VO) method was utilized to establish the CCI rat model, and the CCI rats were identified by the Morris water maze test and histological staining. After the CCI rats were treated with RIPC, the damage to the rat cortex and hippocampal tissues and the status of the ALP were determined. Western blot analysis and immunofluorescence assays were performed to observe the nuclear translocation of TFEB. The rats were injected with TFEB siRNA via the lateral ventricle to investigate the effect of TFEB siRNA on the RIPC-treated CCI rats. The results suggested that RIPC of the CCI rats alleviated nerve injury, induced TFEB translocation into the nucleus, upregulated autophagy-related protein expression, and activated ALP machinery. Furthermore, TFEB siRNA decreased the levels of TFEB and impaired the neuroprotective effects of RIPC on the CCI rats. Collectively, we highlighted that RIPC attenuates damage in CCI rats via the activation of the TFEB-mediated ALP.

Amantadine disrupts lysosomal gene expression: A hypothesis for COVID19 treatment

SARS-coronavirus 2 is the causal agent of the COVID-19 outbreak. SARS-Cov-2 entry into a cell is dependent upon binding of the viral spike (S) protein to cellular receptor and on cleavage of the spike protein by the host cell proteases such as Cathepsin L and Cathepsin B. CTSL/B are crucial elements of lysosomal pathway and both enzymes are almost exclusively located in the lysosomes. CTSL disruption offers potential for CoVID-19 therapies. The mechanisms of disruption include: decreasing expression of CTSL, direct inhibition of CTSL activity and affecting the conditions of CTSL environment (increase pH in the lysosomes).We have conducted a high throughput drug screen gene expression analysis to identify compounds that would downregulate the expression of CTSL/CTSB. One of the top significant results shown to downregulate the expression of the CTSL gene is amantadine (10uM). Amantadine was approved by the US Food and Drug Administration in 1968 as a prophylactic agent for influenza and later for Parkinson's disease. It is available as a generic drug.Amantadine in addition to downregulating CTSL appears to further disrupt lysosomal pathway, hence, interfering with the capacity of the virus to replicate. It acts as a lysosomotropic agent altering the CTSL functional environment. We hypothesize that amantadine could decrease the viral load in SARS-CoV-2 positive patients and as such it may serve as a potent therapeutic decreasing the replication and infectivity of the virus likely leading to better clinical outcomes. Clinical studies will be needed to examine the therapeutic utility of amantadine in COVID-19 infection.

Graded regulation of cellular quiescence depth between proliferation and senescence by a lysosomal dimmer switch

The reactivation of quiescent cells to proliferate is fundamental to tissue repair and homeostasis in the body. Often referred to as the G0 state, quiescence is, however, not a uniform state but with graded depth. Shallow quiescent cells exhibit a higher tendency to revert to proliferation than deep quiescent cells, while deep quiescent cells are still fully reversible under physiological conditions, distinct from senescent cells. Cellular mechanisms underlying the control of quiescence depth and the connection between quiescence and senescence are poorly characterized, representing a missing link in our understanding of tissue homeostasis and regeneration. Here we measured transcriptome changes as rat embryonic fibroblasts moved from shallow to deep quiescence over time in the absence of growth signals. We found that lysosomal gene expression was significantly up-regulated in deep quiescence, and partially compensated for gradually reduced autophagy flux. Reducing lysosomal function drove cells progressively deeper into quiescence and eventually into a senescence-like irreversibly arrested state; increasing lysosomal function, by lowering oxidative stress, progressively pushed cells into shallower quiescence. That is, lysosomal function modulates graded quiescence depth between proliferation and senescence as a dimmer switch. Finally, we found that a gene-expression signature developed by comparing deep and shallow quiescence in fibroblasts can correctly classify a wide array of senescent and aging cell types in vitro and in vivo, suggesting that while quiescence is generally considered to protect cells from irreversible arrest of senescence, quiescence deepening likely represents a common transition path from cell proliferation to senescence, related to aging.

Abstract 985: Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format

Abstract Transcription factor EB (TFEB) is a master regulator of lysosomal gene expression and mitochondrial biogenesis. Autophagy is known to act as a double edged sword in cancer. Autophagy acts as a tumor suppressor during the tumorigenesis process but it also helps to sustain the survival of already transformed cancer cells. Lysosomes sit at the end of the autophagy pathway, which is critical to meet the needs of autophagy. Therefore, small molecules that inhibit TFEB-mediated lysosomal biogenesis and autophagic degradation may be helpful for inhibiting cancer cell growth and limiting cancer progression. On the other hand, small molecules that enhance TFEB-mediated lysosomal degradation may be used to prevent tumorigenesis. In order to identify compounds that modulate TFEB translocation, an image-based TFEB translocation assay was developed in a 1536-well plate format with a GFP-TFEB stable AML12 cell line, which is an immortalized mouse hepatocyte cell line. Torin 1, a known TFEB translocation inducer, was used as positive control in the study. Torin 1 increased TFEB nuclear translocation in a concentration dependent manner with an EC50 of 150nM. To optimize the assay condition, various cell densities per well and time courses were tested. To validate this assay, a group of 384 known anticancer compounds were screened at 11 concentrations ranging from 0.8 nM to 46 μM. From this screening, we identified 25 compounds that enhanced TFEB nuclear translocation with EC50s ranging from 2 nM to 12 μM. Several known and novel TFEB inducers were among these compounds. These results demonstrate that this high-content and high-throughput assay can be used to screen large numbers of chemicals and provide a robust in vitro system to identify TFEB agonists and antagonists, which may be used to either prevent (agonists) or treat (antagonist) cancer. Citation Format: Li Zhang, Ruili Huang, Wen Xing Ding, Menghang Xia. Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 985.

YAP/TAZ Inhibition Induces Metabolic and Signaling Rewiring Resulting in Targetable Vulnerabilities in NF2-Deficient Tumor Cells

Merlin/NF2 is a bona fide tumor suppressor whose mutations underlie inherited tumor syndrome neurofibromatosis type 2 (NF2), as well as various sporadic cancers including kidney cancer. Multiple Merlin/NF2 effector pathways including the Hippo-YAP/TAZ pathway have been identified. However, the molecular mechanisms underpinning the growth and survival of NF2-mutant tumors remain poorly understood. Using an inducible orthotopic kidney tumor model, we demonstrate that YAP/TAZ silencing is sufficient to induce regression of pre-established NF2-deficient tumors. Mechanistically, YAP/TAZ depletion diminishes glycolysis-dependent growth and increases mitochondrial respiration and reactive oxygen species (ROS) buildup, resulting in oxidative-stress-induced cell death when challenged by nutrient stress. Furthermore, we identify lysosome-mediated cAMP-PKA/EPAC-dependent activation of RAF-MEK-ERK signaling as a resistance mechanism to YAP/TAZ inhibition. Finally, unbiased analysis of TCGA primary kidney tumor transcriptomes confirms a positive correlation of a YAP/TAZ signature with glycolysis and inverse correlations with oxidative phosphorylation and lysosomal gene expression, supporting the clinical relevance of our findings.

Genetic Analysis Reveals AMPK Is Required to Support Tumor Growth in Murine Kras-Dependent Lung Cancer Models

AMPK, a conserved sensor of low cellular energy, can either repress or promote tumor growth depending on the context. However, no studies have examined AMPK function in autochthonous genetic mouse models of epithelial cancer. Here, we examine the role of AMPK in murine KrasG12D-mediated non-small-cell lung cancer (NSCLC), a cancer type in humans that harbors frequent inactivating mutations in the LKB1 tumor suppressor-the predominant upstream activating kinase of AMPK and 12 related kinases. Unlike LKB1 deletion, AMPK deletion in KrasG12D lung tumors did not accelerate lung tumor growth. Moreover, deletion of AMPK in KrasG12D p53f/f tumors reduced lung tumor burden. We identified a critical role for AMPK in regulating lysosomal gene expression through the Tfe3 transcription factor, which was required to support NSCLC growth. Thus, AMPK supports the growth of KrasG12D-dependent lung cancer through the induction of lysosomes, highlighting an unrecognized liability of NSCLC.

Lysosomal Cathepsin Protease Gene Expression Profiles in the Human Brain During Normal Development.

Cathepsin protease genes are necessary for protein homeostasis in normal brain development and function. The diversity of the 15 cathepsin protease activities raises the question of what are the human brain expression profiles of the cathepsin genes during development from prenatal and infancy to childhood, adolescence, and young adult stages. This study, therefore, evaluated the cathepsin gene expression profiles in 16 human brain regions during development by quantitative RNA-sequencing data obtained from the Allen Brain Atlas resource. Total expression of all cathepsin genes was the lowest at the early prenatal stage which became increased at the infancy stage. During infancy to young adult phases, total gene expression was similar. Interestingly, the rank ordering of gene expression among the cathepsins was similar throughout the brain at the age periods examined, showing (a) high expression of cathepsins B, D, and F; (b) moderate expression of cathepsins A, L, and Z; (c) low expression of cathepsins C, H, K, O, S, and V; and (d) very low expression of cathepsins E, G, and W. Results show that the human brain utilizes well-defined, balanced patterns of cathepsin gene expression throughout the different stages of human brain development. Knowledge gained by this study of the gene expression profiles of lysosomal cathepsin proteases among human brain regions during normal development is important for advancing future investigations of how these cathepsins are dysregulated in lysosomal-related brain disorders that affect infants, children, adolescents, and young adults.

A novel autophagy enhancer as a therapeutic agent against metabolic syndrome and diabetes

Autophagy is a critical regulator of cellular homeostasis, dysregulation of which is associated with diverse diseases. Here we show therapeutic effects of a novel autophagy enhancer identified by high-throughput screening of a chemical library against metabolic syndrome. An autophagy enhancer increases LC3-I to LC3-II conversion without mTOR inhibition. MSL, an autophagy enhancer, activates calcineurin, and induces dephosphorylation/nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy gene expression. MSL accelerates intracellular lipid clearance, which is reversed by lalistat 2 or Tfeb knockout. Its administration improves the metabolic profile of ob/ob mice and ameliorates inflammasome activation. A chemically modified MSL with increased microsomal stability improves the glucose profile not only of ob/ob mice but also of mice with diet-induced obesity. Our data indicate that our novel autophagy enhancer could be a new drug candidate for diabetes or metabolic syndrome with lipid overload.

PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation

Defects in the PEX5 gene impair the import of peroxisomal matrix proteins, leading to nonfunctional peroxisomes and other associated pathological defects such as Zellweger syndrome. Although PEX5 regulates autophagy process in a stress condition, the mechanisms controlling autophagy by PEX5 under nutrient deprivation are largely unknown. Herein, we show a novel function of PEX5 in the regulation of autophagy via Transcription Factor EB (TFEB). Under serum-starved conditions, when PEX5 is depleted, the mammalian target of rapamycin (mTORC1) inhibitor TSC2 is downregulated, which results in increased phosphorylation of the mTORC1 substrates, including 70S6K, S6K, and 4E-BP-1. mTORC1 activation further suppresses the nuclear localization of TFEB, as indicated by decreased mRNA levels of TFEB, LIPA, and LAMP1. Interestingly, peroxisomal mRNA and protein levels are also reduced by TFEB inactivation, indicating that TFEB might control peroxisome biogenesis at a transcriptional level. Conversely, pharmacological inhibition of mTOR resulting from PEX5 depletion during nutrient starvation activates TFEB by promoting nuclear localization of the protein. In addition, mTORC1 inhibition recovers the damaged-peroxisome biogenesis. These data suggest that PEX5 may be a critical regulator of lysosomal gene expression and autophagy through the mTOR-TFEB-autophagy axis under nutrient deprivation.

Adaptor protein p62 promotes skin tumor growth and metastasis and is induced by UVA radiation

Skin cancer is the most common cancer, and exposure to ultraviolet (UV) radiation, namely UVA and UVB, is the major risk factor for skin cancer development. UVA is significantly less effective in causing direct DNA damage than UVB, but UVA has been shown to increase skin cancer risk. The mechanism by which UVA contributes to skin cancer remains unclear. Here, using RNA-Seq, we show that UVA induces autophagy and lysosomal gene expression, including the autophagy receptor and substrate p62. We found that UVA activates transcription factor EB (TFEB), a known regulator of autophagy and lysosomal gene expression, which, in turn, induces p62 transcription. Next, we identified a novel relationship between p62 and cyclooxygenase-2 (COX-2), a prostaglandin synthase critical for skin cancer development. COX-2 expression was up-regulated by UVA-induced p62, suggesting that p62 plays a role in UVA-induced skin cancer. Moreover, we found that p62 stabilizes COX-2 protein through the p62 ubiquitin-associated domain and that p62 regulates prostaglandin E2 production in vitro In a syngeneic squamous cell carcinoma mouse model, p62 knockdown inhibited tumor growth and metastasis. Furthermore, p62-deficient tumors exhibited reduced immune cell infiltration and increased cell differentiation. Because prostaglandin E2 is known to promote pro-tumorigenic immune cell infiltration, increase proliferation, and inhibit keratinocyte differentiation in vivo, this work suggests that UVA-induced p62 acts through COX-2 to promote skin tumor growth and progression. These findings expand our understanding of UVA-induced skin tumorigenesis and tumor progression and suggest that targeting p62 can help prevent or treat UVA-associated skin cancer.