The Experts below are selected from a list of 2946 Experts worldwide ranked by ideXlab platform
Rosa Puertollano - One of the best experts on this subject based on the ideXlab platform.
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the transcription factors tfe3 and TFEB amplify p53 dependent transcriptional programs in response to dna damage
eLife, 2018Co-Authors: Owen A. Brady, Eutteum Jeong, Jose A Martina, Mehdi Pirooznia, Ilker Tunc, Rosa PuertollanoAbstract:: The transcription factors TFE3 and TFEB cooperate to regulate autophagy induction and lysosome biogenesis in response to starvation. Here we demonstrate that DNA damage activates TFE3 and TFEB in a p53 and mTORC1 dependent manner. RNA-Seq analysis of TFEB/TFE3 double-knockout cells exposed to etoposide reveals a profound dysregulation of the DNA damage response, including upstream regulators and downstream p53 targets. TFE3 and TFEB contribute to sustain p53-dependent response by stabilizing p53 protein levels. In TFEB/TFE3 DKOs, p53 half-life is significantly decreased due to elevated Mdm2 levels. Transcriptional profiles of genes involved in lysosome membrane permeabilization and cell death pathways are dysregulated in TFEB/TFE3-depleted cells. Consequently, prolonged DNA damage results in impaired LMP and apoptosis induction. Finally, expression of multiple genes implicated in cell cycle control is altered in TFEB/TFE3 DKOs, revealing a previously unrecognized role of TFEB and TFE3 in the regulation of cell cycle checkpoints in response to stress.
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the transcription factors TFEB and tfe3 link the flcn ampk signaling axis to innate immune response and pathogen resistance
bioRxiv, 2018Co-Authors: Leeanna Elhoujeiri, Jose A Martina, Elite Possik, Tarika Vijayaraghavan, Mathieu Paquette, Jalal M Kazan, Eric H, Russell G Jones, Paola Blanchette, Rosa PuertollanoAbstract:TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK conferred pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, while ablation of total AMPK activity abolished this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induced TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages was observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved and pharmacologically actionable mechanism coupling energy status with innate immunity.
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protein phosphatase 2a stimulates activation of TFEB and tfe3 transcription factors in response to oxidative stress
Journal of Biological Chemistry, 2018Co-Authors: Jose A Martina, Rosa PuertollanoAbstract:: Adaptations and responses to stress conditions are fundamental processes that all cells must accomplish to maintain or restore cellular homeostasis. Cells have a plethora of response pathways to mitigate the effect of different environmental stressors. The transcriptional regulators transcription factor EB (TFEB) and transcription factor binding to IGHM enhancer 3 (TFE3) play a key role in the control of these stress pathways. Therefore, understanding their regulation under different stress conditions is of great interest. Here, using a range of human and murine cells, we show that TFEB and TFE3 are activated upon induction of acute oxidative stress by sodium arsenite via an mTOR complex 1 (mTORC1)-independent process. We found that the mechanism of arsenite-stimulated TFEB and TFE3 activation instead involves protein phosphatase 2A (PP2A)-mediated dephosphorylation at Ser-211 and Ser-321, respectively. Depletion of either the catalytic (PPP2CA+B) or regulatory (PPP2R2A/B55α) subunits of PP2A, as well as PP2A inactivation with the specific inhibitor okadaic acid, abolished TFEB and TFE3 activation in response to sodium arsenite. Conversely, PP2A activation by ceramide or the sphingosine-like compound FTY720 was sufficient to induce TFE3 nuclear translocation. MS analysis revealed that PP2A dephosphorylates TFEB at several residues, including Ser-109, Ser-114, Ser-122, and Ser-211, thus facilitating TFEB activation. Overall, this work identifies a critical mechanism that activates TFEB and TFE3 without turning off mTORC1 activity. We propose that this mechanism may enable some cell types such as immune or cancer cells that require simultaneous TFEB/TFE3 and mTORC1 signaling to survive and achieve robust cell growth in stressful environments.
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the complex relationship between TFEB transcription factor phosphorylation and subcellular localization
The EMBO Journal, 2018Co-Authors: Rosa Puertollano, Shawn M Ferguson, James Brugarolas, Andrea BallabioAbstract:Abstract The MiT‐TFE family of basic helix‐loop‐helix leucine‐zipper transcription factors includes four members: TFEB, TFE3, TFEC, and MITF. Originally described as oncogenes, these factors play a major role as regulators of lysosome biogenesis, cellular energy homeostasis, and autophagy. An important mechanism by which these transcription factors are regulated involves their shuttling between the surface of lysosomes, the cytoplasm, and the nucleus. Such dynamic changes in subcellular localization occur in response to nutrient fluctuations and various forms of cell stress and are mediated by changes in the phosphorylation of multiple conserved amino acids. Major kinases responsible for MiT‐TFE protein phosphorylation include mTOR, ERK, GSK3, and AKT. In addition, calcineurin de‐phosphorylates MiT‐TFE proteins in response to lysosomal calcium release. Thus, through changes in the phosphorylation state of MiT‐TFE proteins, lysosome function is coordinated with the cellular metabolic state and cellular demands. This review summarizes the evidence supporting MiT‐TFE regulation by phosphorylation at multiple key sites. Elucidation of such regulatory mechanisms is of fundamental importance to understand how these transcription factors contribute to both health and disease.
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emerging roles for TFEB in the immune response and inflammation
Autophagy, 2018Co-Authors: Owen A. Brady, Jose A Martina, Rosa PuertollanoAbstract:ABSTRACTInflammation is a central feature of an effective immune response, which functions to eliminate pathogens and other foreign material, and promote recovery; however, dysregulation of the inflammatory response is associated with a wide variety of disease states. The autophagy-lysosome pathway is one of 2 major degradative pathways used by the cell and serves to eliminate long-lived and dysfunctional proteins and organelles to maintain homeostasis. Mounting evidence implicates the autophagy-lysosome pathway as a key player in regulating the inflammatory response; hence many inflammatory diseases may fundamentally be diseases of autophagy-lysosome pathway dysfunction. The recent identification of TFEB and TFE3 as master regulators of macroautophagy/autophagy and lysosome function raises the possibility that these transcription factors may be of central importance in linking autophagy and lysosome dysfunction with inflammatory disorders. Here, we review the current state of knowledge linking TFEB and T...
Andrea Ballabio - One of the best experts on this subject based on the ideXlab platform.
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TFEB Modulates p21/WAF1/CIP1 during the DNA Damage Response.
Cells, 2020Co-Authors: Sandra Pisonero-vaquero, Andrea Ballabio, Chiara Soldati, Marcella Cesana, Diego L. MedinaAbstract:: The MiT/TFE family of transcription factors (MITF, TFE3, and TFEB), which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents.
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TFEB driven endocytosis coordinates mtorc1 signaling and autophagy
Autophagy, 2019Co-Authors: Israel C Nnah, Diego L. Medina, Gennaro Napolitano, Biao Wang, Chaitali Saqcena, Gregory F Weber, Edward M Bonder, Dustin Bagley, Rossella De Cegli, Andrea BallabioAbstract:ABSTRACTThe mechanistic target of rapamycin kinase complex 1 (MTORC1) is a central cellular kinase that integrates major signaling pathways, allowing for regulation of anabolic and catabolic processes including macroautophagy/autophagy and lysosomal biogenesis. Essential to these processes is the regulatory activity of TFEB (transcription factor EB). In a regulatory feedback loop modulating transcriptional levels of RRAG/Rag GTPases, TFEB controls MTORC1 tethering to membranes and induction of anabolic processes upon nutrient replenishment. We now show that TFEB promotes expression of endocytic genes and increases rates of cellular endocytosis during homeostatic baseline and starvation conditions. TFEB-mediated endocytosis drives assembly of the MTORC1-containing nutrient sensing complex through the formation of endosomes that carry the associated proteins RRAGD, the amino acid transporter SLC38A9, and activate AKT/protein kinase B (AKT p-T308). TFEB-induced signaling endosomes en route to lysosomes are i...
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overexpression of TFEB drives a pleiotropic neurotrophic effect and prevents parkinson s disease related neurodegeneration
Molecular Therapy, 2018Co-Authors: Albert Torra, Andrea Ballabio, Annabelle Parent, Thais Cuadros, Beatriz Rodriguezgalvan, Esther Ruizbronchal, Analia Bortolozzi, Miquel Vila, Jordi BoveAbstract:The possible implication of transcription factor EB (TFEB) as a therapeutic target in Parkinson's disease has gained momentum since it was discovered that TFEB controls lysosomal biogenesis and autophagy and that its activation might counteract lysosomal impairment and protein aggregation. However, the majority of putative direct targets of TFEB described to date is linked to a range of biological processes that are not related to the lysosomal-autophagic system. Here, we assessed the effect of overexpressing TFEB with an adeno-associated viral vector in mouse substantia nigra dopaminergic neurons. We demonstrate that TFEB overexpression drives a previously unknown bona fide neurotrophic effect, giving rise to cell growth, higher tyrosine hydroxylase levels, and increased dopamine release in the striatum. TFEB overexpression induces the activation of the mitogen-activated protein kinase 1/3 (MAPK1/3) and AKT pro-survival pathways, phosphorylation of mTORC1 effectors 4E-binding protein 1 (4E-BP1) and S6 kinase B1 (S6K1), and increased protein synthesis. We show that TFEB overexpression prevents dopaminergic cell loss and counteracts atrophy and the associated protein synthesis decline in the MPTP mouse model of Parkinson's disease. Our results suggest that increasing TFEB activity might prevent neuronal death and restore neuronal function in Parkinson's disease and other neurodegenerative diseases through different mechanisms.
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the complex relationship between TFEB transcription factor phosphorylation and subcellular localization
The EMBO Journal, 2018Co-Authors: Rosa Puertollano, Shawn M Ferguson, James Brugarolas, Andrea BallabioAbstract:Abstract The MiT‐TFE family of basic helix‐loop‐helix leucine‐zipper transcription factors includes four members: TFEB, TFE3, TFEC, and MITF. Originally described as oncogenes, these factors play a major role as regulators of lysosome biogenesis, cellular energy homeostasis, and autophagy. An important mechanism by which these transcription factors are regulated involves their shuttling between the surface of lysosomes, the cytoplasm, and the nucleus. Such dynamic changes in subcellular localization occur in response to nutrient fluctuations and various forms of cell stress and are mediated by changes in the phosphorylation of multiple conserved amino acids. Major kinases responsible for MiT‐TFE protein phosphorylation include mTOR, ERK, GSK3, and AKT. In addition, calcineurin de‐phosphorylates MiT‐TFE proteins in response to lysosomal calcium release. Thus, through changes in the phosphorylation state of MiT‐TFE proteins, lysosome function is coordinated with the cellular metabolic state and cellular demands. This review summarizes the evidence supporting MiT‐TFE regulation by phosphorylation at multiple key sites. Elucidation of such regulatory mechanisms is of fundamental importance to understand how these transcription factors contribute to both health and disease.
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stub1 regulates TFEB induced autophagy lysosome pathway
The EMBO Journal, 2017Co-Authors: Carmine Settembre, Andrea Ballabio, Tony N EissaAbstract:Abstract TFEB is a master regulator for transcription of genes involved in autophagy and lysosome biogenesis. Activity of TFEB is inhibited upon its serine phosphorylation by mTOR. The overall mechanisms by which TFEB activity in the cell is regulated are not well elucidated. Specifically, the mechanisms of TFEB turnover and how they might influence its activity remain unknown. Here, we show that STUB1, a chaperone‐dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin–proteasome pathway. Phosphorylated TFEB accumulated in STUB1‐deficient cells and in tissues of STUB1‐deficient mice resulting in reduced TFEB activity. Conversely, cellular overexpression of STUB1 resulted in reduced phosphorylated TFEB and increased TFEB activity. STUB1 preferentially interacted with and ubiqutinated phosphorylated TFEB, targeting it to proteasomal degradation. Consistent with reduced TFEB activity, accumulation of phosphorylated TFEB in STUB1‐deficient cells resulted in reduced autophagy and reduced mitochondrial biogenesis. These studies reveal that the ubiquitin–proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.
Pedram Argani - One of the best experts on this subject based on the ideXlab platform.
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TFEB expression profiling in renal cell carcinomas clinicopathologic correlations
The American Journal of Surgical Pathology, 2019Co-Authors: Sounak Gupta, Pedram Argani, Yingbei Chen, Achim A Jungbluth, Satish K Tickoo, Samson W Fine, Anuradha Gopalan, Hikmat Alahmadie, Sahussapont Joseph Sirintrapun, Alejandro SanchezAbstract:TFEB is overexpressed in TFEB-rearranged renal cell carcinomas as well as in renal tumors with amplifications of TFEB at 6p21.1. As recent literature suggests that renal tumors with 6p21.1 amplification behave more aggressively than those with rearrangements of TFEB, we compared relative TFEB gene e
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vegfa amplification increased gene copy number and vegfa mrna expression in renal cell carcinoma with TFEB gene alterations
Modern Pathology, 2019Co-Authors: Anna Calio, Matteo Brunelli, Diego Segala, Pedram Argani, Serena Pedron, Claudio Doglioni, Guido MartignoniAbstract:Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3–4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.
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VEGFA amplification/increased gene copy number and VEGFA mRNA expression in renal cell carcinoma with TFEB gene alterations.
Modern Pathology, 2018Co-Authors: Anna Calio, Matteo Brunelli, Diego Segala, Pedram Argani, Serena Pedron, Claudio Doglioni, Guido MartignoniAbstract:Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3–4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.
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TFEB amplified renal cell carcinomas an aggressive molecular subset demonstrating variable melanocytic marker expression and morphologic heterogeneity
The American Journal of Surgical Pathology, 2016Co-Authors: Pedram Argani, George J Netto, Victor E Reuter, Lei Zhang, Yun Shao Sung, Yi Ning, Jonathan I Epstein, Cristina R AntonescuAbstract:Abstract Renal cell carcinomas (RCCs) with the t(6;11)(p21;q12) chromosome translocation are low-grade RCC which often occur in young patients. They typically feature an unusual biphasic morphology characterized by nests of larger epithelioid cells surrounding intraluminal collections of smaller cells clustered around basement membrane material. The t(6;11)(p21;q12) translocation fuses the Alpha (MALAT1) gene with the TFEB transcription factor gene, resulting in upregulated expression of intact native TFEB that drives the aberrant expression of melanocytic markers which is a hallmark of this distinctive neoplasm. We now report 8 cases of RCC, which demonstrate TFEB gene amplification (6 without TFEB rearrangement, 2 with concurrent TFEB rearrangement) and demonstrate downstream consequences of TFEB overexpression. Like the unamplified t(6;11) RCC, all TFEB-amplified RCC were associated with aberrant melanocytic marker expression. However, several differences between TFEB-amplified RCC and the usual unamplified t(6;11) RCC are evident. First, TFEB-amplified RCC occurred in older patients (median age, 64.5 y) compared with unamplified t(6;11) RCC (median age, 31 y). Second, the morphology of TFEB-amplified RCC is not entirely distinctive, frequently featuring nests of high-grade epithelioid cells with eosinophilic cytoplasm associated with pseudopapillary formation and necrosis, or true papillary formations. These patterns raise the differential diagnosis of high-grade clear cell and papillary RCC. Third, TFEB and melanocytic marker expression was more variable within the TFEB-amplified RCC. TFEB protein expression by immunohistochemistry was detectable in 6 of 8 cases. While all 8 cases expressed melan-A, only 5 of 8 expressed cathepsin K and only 3 of 8 expressed HMB45. Fourth, the TFEB-amplified RCC were associated with a more aggressive clinical course; 3 of 8 cases presented with advanced stage or metastatic disease, 2 subsequently developed metastatic disease, whereas the other 3 cases had minimal/no follow-up. Our results are corroborated by scant data reported on 6 TFEB-amplified RCC in the literature, gleaned from 1 case report, 1 abstract, and 4 individual cases identified within 2 genomic studies of large cohorts of RCC. In summary, TFEB-amplified RCC represent a distinct molecular subtype of high-grade adult RCC associated with aggressive clinical behavior, variable morphology, and aberrant melanocytic marker expression.
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molecular confirmation of t 6 11 p21 q12 renal cell carcinoma in archival paraffin embedded material using a break apart TFEB fish assay expands its clinicopathologic spectrum
The American Journal of Surgical Pathology, 2012Co-Authors: Pedram Argani, Peter B. Illei, Marc Ladanyi, Raluca Yonescu, Laura Morsberger, Kerry Morris, George J Netto, Nathan Smith, Nilda Gonzalez, Constance A GriffinAbstract:The past decade has witnessed the characterization of a subset of renal cell carcinomas (RCCs) that have chromosomal translocations resulting in gene fusions involving members of the MiT subfamily of transcription factors. The best known members of this subset are the Xp11 translocation RCCs, which were recognized by the World Health Organization in 2004.1 These neoplasms comprise the majority of pediatric RCCs and a smaller percentage of adult RCCs and classically feature a papillary architecture lined by clear cells with extensive psammomatous calcification.2–8 Xp11 translocation RCCs are characterized by gene fusions involving the TFE3 transcription factor gene that maps to this locus; at least 5 different fusion partners for TFE3 have been identified to date.2,3,5,9,10 A less well-known member of the translocation RCC family is the subset of RCCs characterized by t(6;11)(p21;q12), which results in fusion of the untranslated Alpha (MALAT1) gene on 11q12 to the related TFEB gene on 6p21.11–14 Only 21 genetically confirmed cases of t(6;11) RCCs have been reported.5,11–23 This neoplasm typically demonstrates a distinctive biphasic morphology, comprising larger epithelioid cells and smaller cells clustered around basement membrane material; however, the full spectrum of its morphologic appearances is not known. The t(6;11) RCCs differ from most conventional RCCs in that they consistently express melanocytic immunohistochemical (IHC) markers such as HMB45, Melan A, and the cysteine protease cathepsin K24,25 but are either negative or only focally positive for epithelial markers such as cytokeratins.11,12 On the basis of clinical, pathologic, and genetic similarities between the t(6;11) RCCs and the Xp11 translocation RCCs, we have proposed that these 2 neoplasms be classified together under the broader category of “MiT family translocation RCC.”12 Molecular confirmation of a diagnosis of a translocation RCC is relatively simple if fresh tissue is available for either cytogenetics or reverse transcriptase polymerase chain reaction assay using primers from the genes known to be involved in the gene fusion. However, in many cases, only archival, formalin-fixed, paraffin-embedded material is available. For the Xp11 translocation RCCs and t(6;11) RCCs, IHC for TFE3 and TFEB, respectively, have proven to be useful for confirming the diagnosis in archival material.6,12 This is because both TFE3 fusion proteins and native TFEB are upregulated by promoter substitution by the gene fusions in these 2 RCCs relative to the level of expression of the respective native proteins. However, IHC is highly fixation dependent and has proven to be particularly difficult for TFE3 and TFEB for several reasons. These include the scarcity of genetically confirmed positive controls and the fact that the assays are optimally performed by overnight incubation, which is difficult to automate.26 Recently, break-apart fluorescence in situ hybridization (FISH) assays for TFE3 gene fusions were developed for archival material27–29 and have allowed the expansion of the morphologic spectrum of the Xp11 translocation RCCs.30 A validated FISH assay for molecular confirmation of t(6;11) RCC has not been reported previously. We report herein the development of a break-apart TFEB FISH assay for the diagnosis of t(6;11)(p21;q12) RCCs. We validated the assay on 4 genetically confirmed cases and 76 pertinent negative control cases, confirmed the presence of a TFEB gene rearrangement in a previously reported TFEB IHC-positive case from 46 years ago, and used the assay to report 8 new cases that expanded the clinicopathologic spectrum of t(6;11) RCCs.
Jose A Martina - One of the best experts on this subject based on the ideXlab platform.
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the transcription factors tfe3 and TFEB amplify p53 dependent transcriptional programs in response to dna damage
eLife, 2018Co-Authors: Owen A. Brady, Eutteum Jeong, Jose A Martina, Mehdi Pirooznia, Ilker Tunc, Rosa PuertollanoAbstract:: The transcription factors TFE3 and TFEB cooperate to regulate autophagy induction and lysosome biogenesis in response to starvation. Here we demonstrate that DNA damage activates TFE3 and TFEB in a p53 and mTORC1 dependent manner. RNA-Seq analysis of TFEB/TFE3 double-knockout cells exposed to etoposide reveals a profound dysregulation of the DNA damage response, including upstream regulators and downstream p53 targets. TFE3 and TFEB contribute to sustain p53-dependent response by stabilizing p53 protein levels. In TFEB/TFE3 DKOs, p53 half-life is significantly decreased due to elevated Mdm2 levels. Transcriptional profiles of genes involved in lysosome membrane permeabilization and cell death pathways are dysregulated in TFEB/TFE3-depleted cells. Consequently, prolonged DNA damage results in impaired LMP and apoptosis induction. Finally, expression of multiple genes implicated in cell cycle control is altered in TFEB/TFE3 DKOs, revealing a previously unrecognized role of TFEB and TFE3 in the regulation of cell cycle checkpoints in response to stress.
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the transcription factors TFEB and tfe3 link the flcn ampk signaling axis to innate immune response and pathogen resistance
bioRxiv, 2018Co-Authors: Leeanna Elhoujeiri, Jose A Martina, Elite Possik, Tarika Vijayaraghavan, Mathieu Paquette, Jalal M Kazan, Eric H, Russell G Jones, Paola Blanchette, Rosa PuertollanoAbstract:TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK conferred pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, while ablation of total AMPK activity abolished this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induced TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages was observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved and pharmacologically actionable mechanism coupling energy status with innate immunity.
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protein phosphatase 2a stimulates activation of TFEB and tfe3 transcription factors in response to oxidative stress
Journal of Biological Chemistry, 2018Co-Authors: Jose A Martina, Rosa PuertollanoAbstract:: Adaptations and responses to stress conditions are fundamental processes that all cells must accomplish to maintain or restore cellular homeostasis. Cells have a plethora of response pathways to mitigate the effect of different environmental stressors. The transcriptional regulators transcription factor EB (TFEB) and transcription factor binding to IGHM enhancer 3 (TFE3) play a key role in the control of these stress pathways. Therefore, understanding their regulation under different stress conditions is of great interest. Here, using a range of human and murine cells, we show that TFEB and TFE3 are activated upon induction of acute oxidative stress by sodium arsenite via an mTOR complex 1 (mTORC1)-independent process. We found that the mechanism of arsenite-stimulated TFEB and TFE3 activation instead involves protein phosphatase 2A (PP2A)-mediated dephosphorylation at Ser-211 and Ser-321, respectively. Depletion of either the catalytic (PPP2CA+B) or regulatory (PPP2R2A/B55α) subunits of PP2A, as well as PP2A inactivation with the specific inhibitor okadaic acid, abolished TFEB and TFE3 activation in response to sodium arsenite. Conversely, PP2A activation by ceramide or the sphingosine-like compound FTY720 was sufficient to induce TFE3 nuclear translocation. MS analysis revealed that PP2A dephosphorylates TFEB at several residues, including Ser-109, Ser-114, Ser-122, and Ser-211, thus facilitating TFEB activation. Overall, this work identifies a critical mechanism that activates TFEB and TFE3 without turning off mTORC1 activity. We propose that this mechanism may enable some cell types such as immune or cancer cells that require simultaneous TFEB/TFE3 and mTORC1 signaling to survive and achieve robust cell growth in stressful environments.
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emerging roles for TFEB in the immune response and inflammation
Autophagy, 2018Co-Authors: Owen A. Brady, Jose A Martina, Rosa PuertollanoAbstract:ABSTRACTInflammation is a central feature of an effective immune response, which functions to eliminate pathogens and other foreign material, and promote recovery; however, dysregulation of the inflammatory response is associated with a wide variety of disease states. The autophagy-lysosome pathway is one of 2 major degradative pathways used by the cell and serves to eliminate long-lived and dysfunctional proteins and organelles to maintain homeostasis. Mounting evidence implicates the autophagy-lysosome pathway as a key player in regulating the inflammatory response; hence many inflammatory diseases may fundamentally be diseases of autophagy-lysosome pathway dysfunction. The recent identification of TFEB and TFE3 as master regulators of macroautophagy/autophagy and lysosome function raises the possibility that these transcription factors may be of central importance in linking autophagy and lysosome dysfunction with inflammatory disorders. Here, we review the current state of knowledge linking TFEB and T...
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TFEB regulates lysosomal positioning by modulating tmem55b expression and jip4 recruitment to lysosomes
Nature Communications, 2017Co-Authors: Rose Willett, Jose A Martina, James P Zewe, Rachel C Wills, Gerald R V Hammond, Rosa PuertollanoAbstract:Lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. Here we report the identification of a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome–lysosome fusion. Overall our data suggest that the TFEB/TMEM55B/JIP4 pathway coordinates lysosome movement in response to a variety of stress conditions. Lysosomal distribution is linked to the role of lysosomes in many cellular functions. Here the authors show that the lysosomal protein TMEM55B is regulated by TFEB and recruits JIP4 to the lysosomal surface inducing dynein-dependent transport of lysosomes toward the cell center in response to stress conditions.
Guido Martignoni - One of the best experts on this subject based on the ideXlab platform.
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vegfa amplification increased gene copy number and vegfa mrna expression in renal cell carcinoma with TFEB gene alterations
Modern Pathology, 2019Co-Authors: Anna Calio, Matteo Brunelli, Diego Segala, Pedram Argani, Serena Pedron, Claudio Doglioni, Guido MartignoniAbstract:Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3–4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.
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VEGFA amplification/increased gene copy number and VEGFA mRNA expression in renal cell carcinoma with TFEB gene alterations.
Modern Pathology, 2018Co-Authors: Anna Calio, Matteo Brunelli, Diego Segala, Pedram Argani, Serena Pedron, Claudio Doglioni, Guido MartignoniAbstract:Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3–4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.
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perivascular epithelioid cell tumors pecomas harboring tfe3 gene rearrangements lack the tsc2 alterations characteristic of conventional pecomas further evidence for a biological distinction
The American Journal of Surgical Pathology, 2012Co-Authors: Izabela A Malinowska, Guido Martignoni, George J Netto, David J Kwiatkowski, Sharon W Weiss, Pedram ArganiAbstract:Perivascular epithelioid cell neoplasms (PEComas) are a group of lesions composed of distinctive perivascular epithelioid cells which typically demonstrate both melanocytic and muscular differentiation. This family includes the common renal angiomyolipoma, pulmonary clear cell sugar tumor, lymphangioleiomyomatosis, and less common neoplasms of the soft tissue, gynecologic and gastrointestinal tracts (7, 8, 9, 14, 16, 19). The cells comprising these lesions may be variably epithelioid or spindled in shape, and have variable cytoplasm ranging from clear to eosinophilic. By immunohistochemistry (IHC), PEComas typically express the melanocytic markers HMB45 and Melan-A and the protease Cathepsin K (18), but also typically label for smooth muscle actin and may express desmin. Some members of the PEComa family (specifically angiomyolipoma and lymphangioleiomyomatosis) are seen with high frequency in the genetic syndrome Tuberous Sclerosis Complex (TSC) (16), and a high frequency of syndromic and sporadic PEComas have demonstrated inactivation of the TSC1 or TSC2 genes (12, 20, 21) with subsequent activation of the mammalian target of rapamycin (mTOR) pathway (15). Specifically, mutation in and loss of heterozygosity (LOH) of TSC2 with loss of expression of the protein tuberin, the protein encoded by TSC2, is consistently found in conventional PEComas. TFE3 is a member of the MiT family of transcription factors, which includes MiTF, TFEB, TFEC, and TFE3 (11). TFE3 gene fusions are known to occur in several types of neoplasia. Alveolar soft part sarcoma (ASPS), a rare epithelioid cell soft tissue sarcoma of uncertain histogenesis, characteristically demonstrates a der (17) t(X;17)(p11;q25) resulting in an ASPL-TFE3 gene fusion (17). In addition, a group of recently-described renal cell carcinomas (RCCs) which often occur in children bear various TFE3 gene fusions; these are designated the Xp11 translocation RCC (1, 2, 5, 6). Moreover, a distinctive subgroup of renal cancers in young patients with overlapping features of melanoma, RCC and PEComas have also proven to harbor TFE3 gene fusions (3). Finally, we have recently identified a subgroup of lesions currently characterized as PEComas which, in contrast to conventional PEComas, harbor TFE3 gene fusions (4). Although the number of cases identified are small, distinctive features of these TFE3-rearranged PEComas include a tendency to young age, absence of the association with tuberous sclerosis, predominant alveolar architecture and epithelioid cytology, minimal immunoreactivity for muscle markers, and strong (3+) TFE3 immunoreactivity. In contrast, conventional PEComas frequently have a spindle cell component, typically label for muscle markers, lack strong TFE3 immunoreactivity, and in young patients are frequently associated with tuberous sclerosis. Since conventional PEComas frequently demonstrate TSC2 LOH, and loss of expression of the tuberin protein which this gene encodes (13), we evaluated TFE3-rearranged PEComas for TSC2 LOH and for tuberin expression by IHC. The study cohort consisted of four PEComas previously shown to harbor TFE3 gene fusions (4), and four conventional PEComas which lacked TFE3 alterations. To assess LOH or allelic loss we performed analysis of three microsatellite markers, STR3, KG8 and STR7 in the region of the TSC2 gene, on paraffin-extracted DNA from tumor and normal tissue as described elsewhere (21). IHC was performed by standard techniques using Target Retrieval Solution pH 6.1 (Dako), incubation with anti-tuberin antibody (1:200 dilution, Cell Signaling, #4308), and development with horseradish peroxidase (HRP)-conjugated secondary antibody and DAB (Dako Envision System). Slides were counterstained with hematoxylin. By IHC, all four of the conventional non-TFE3 PEComas demonstrated loss of tuberin protein labeling by immunohistochemistry, with the surrounding normal tissue serving as an internal control (Figure 1, top row). In contrast, all four of the PEComas previously shown to harbor TFE3 gene fusions demonstrated intact, robust tuberin protein labeling (Figure 1, bottom row). In addition, two of the four conventional PEComas showed LOH or allelic loss for one or more TSC2 microsatellite markers (Figure 1, top right), as we and others have seen previously (12, 15, 20). In contrast, none of the four PEComas previously shown to harbor TFE3 gene fusions demonstrated TSC2 LOH (Figure 1, bottom right). Figure1 Hematoxylin and Eosin staining, IHC for tuberin, and TSC2 microsatellite marker analysis is shown for a conventional PEComa (top row), and a TFE3-rearranged PEComa (bottom row). The insets show regions of normal tissue stained for TSC2 from each tumor ... Thus, these observations, while limited in scope due to the limited number of cases available to us, are consistent with our hypothesis that there is a different pathogenetic mechanism in TFE3-rearranged PEComas which does not involve the TSC2 gene through mutation or allelic loss, or other mechanisms of loss of expression. Thus, they suggest that TFE3-rearranged PEComas represent an entity which morphologically overlaps with conventional PEComas, but is biologically distinctive. This concept has clinical translational importance in that mTORC1 inhibitors, such as rapamycin and everolimus, have been shown to be effective in some cases of PEComas (22). If there is no TSC2 gene involvement in TFE3-rearranged PEComas, then these patients may not respond to mTORC1 inhibitors.
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differential expression of cathepsin k in neoplasms harboring tfe3 gene fusions
Modern Pathology, 2011Co-Authors: Guido Martignoni, Georges J Netto, Philippe Camparo, Peter B. Illei, Franco Bonetti, Stefano Gobbo, Matteo Brunelli, Diego Segala, Enrico Munari, Marc LadanyiAbstract:Cathepsin K is a protease whose expression is driven by microphthalmia transcription factor (MITF) in osteoclasts. TFE3 and TFEB are members of the same transcription factor subfamily as MITF and all three have overlapping transcriptional targets. We have shown that all t(6;11) renal cell carcinomas, which harbor an Alpha-TFEB gene fusion, as well as a subset of the Xp11 translocation renal carcinomas, which harbor various TFE3 gene fusions, express cathepsin K, while no other common renal carcinoma does. We have hypothesized that overexpression of TFEB or certain TFE3 fusion proteins function like MITF in these neoplasms, and thus activate cathepsin K expression. However, the expression of cathepsin K in specific genetic subtypes of Xp11 translocation carcinomas, as well as alveolar soft part sarcoma, which harbors the same ASPSCR1-TFE3 gene fusion as some Xp11 translocation carcinomas, has not been addressed. We performed immunohistochemistry for cathepsin K on 14 genetically confirmed t(X;1)(p11;q21) carcinomas, harboring the PRCC-TFE3 gene fusion; eight genetically confirmed t(X;17)(p11;q25) carcinomas, harboring the ASPSCR1-TFE3 gene fusion; and 18 alveolar soft part sarcomas (12 genetically confirmed), harboring the identical ASPSCR1-TFE3 gene fusion. All 18 alveolar soft part sarcomas expressed cathepsin K. In contrast, all eight ASPSCR1-TFE3 carcinomas were completely negative for cathepsin K. However, 12 of 14 PRCC-TFE3 carcinomas expressed cathepsin K. Expression of cathepsin K distinguishes alveolar soft part sarcoma from the ASPSCR1-TFE3 carcinoma, harboring the same gene fusion. The latter can be useful diagnostically, especially when alveolar soft part sarcoma presents in an unusual site (such as bone) or with clear cell morphology, which raises the differential diagnosis of metastatic ASPSCR1-TFE3 renal cell carcinoma. The difference in expression of cathepsin K between the PRCC-TFE3 and ASPSCR1-TFE3 carcinomas, together with the observed clinical differences between these subtypes of Xp11 translocation carcinomas, suggests the possibility of functional differences between these two related fusion proteins.
