Autophagy in Model Organisms: Insights into Cancer

Autophagy is an evolutionarily conserved process utilized by most organisms to clear cellular damage and recycle building blocks for energy production. In this chapter, we emphasize the importance of genetic model organisms, including yeast, nematodes, flies, and mammals in the discovery and understanding of the autophagy process. We highlight the important roles of autophagy in aging, stress tolerance, neuronal health, organismal development, and pathogen resistance in invertebrate and vertebrate model organisms. We provide examples on how the same autophagy‐related pathways that increase stress response and longevity in lower organisms could be utilized by cancer cells to survive harsh microenvironments, proliferate, and metastasize, with emphasis on the dual role of autophagy in cancer: an antitumorigenic or a protumorigenic process

Characterization of the role of the tumor suppressor FLCN using «Caenorhabditis elegans» and mammalian cells

Birt-Hogg-Dubé is a dominantly-inherited syndrome that increases predisposition to cancer mainly renal tumors and cysts. Folliculin (FLCN), a protein highly conserved across evolution, is the tumor suppressor responsible for this disease. Despite the intensive research effort spent since its discovery in 2001, the cellular role of FLCN remains unclear and how its loss leads to tumorigenesis is not yet defined. FLCN is a binding partner of the 5'AMP-activated protein kinase (AMPK), a central regulator of energy homeostasis. However, the genetic and functional links as well as the phenotypic outcomes on cells/organisms have not been established. In this thesis, we investigated the role of FLCN in the model organism Caenorhabditis elegans (C. elegans) and in mammalian cells. In the first part, we demonstrate that FLCN is an evolutionary conserved negative regulator of AMPK. Loss of FLCN in worms and mammals activates AMPK signaling and induces autophagy, improving cellular bioenergetics, and leading to an advantageous resistance to several metabolic stresses including oxidative stress, anoxia, heat, and nutrient deprivation. In the second part, we highlight the discovery of a novel function for FLCN/AMPK in the regulation of glycogen metabolism and resistance to hyperosmotic stress in C. elegans. We show that loss of flcn-1, leads to AMPK-dependent glycogen accumulation and resistance to hyperosmotic conditions. Upon exposure to salt stress, glycogen reserves are rapidly degraded, leading to the accumulation of the organic osmolyte glycerol, which is crucial for organismal survival to hyperosmotic environments. Importantly, we also show that that the regulation of glycogen metabolism by FLCN is evolutionary conserved and that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from BHD patients. Results of this part demonstrate a dual role for glycogen reserves: an energy reservoir and a store that fuels osmolyte production. In the third part, we investigate the transcriptional regulation downstream FLCN-1 in C. elegans. The results of this chapter indicate that the transcriptional profiles upon loss flcn-1 at basal level significantly overlap with published stress response signatures including oxidative stress, hyperosmotic stress, and infection with pathogens. Furthermore, we found that loss of flcn-1 in C. elegans leads to increased resistance to pathogens impinging on a possible role for FLCN-1 in the regulation of innate immunity. Finally, we found that many stress response genes upregulated in flcn-1 animals are downregulated in hlh-30 mutant animals, and that the hyperosmotic stress resistance in flcn-1 nematodes is abolished upon loss of hlh-30, supporting a potential role of the TFEB worm homolog, HLH-30, in stress response downstream FLCN-1. Altogether, these studies have established FLCN as an evolutionary conserved negative regulator of AMPK, and have led to the discovery of two distinct pathways of stress resistance downstream FLCN/AMPK, a pathway of resistance to metabolic stresses and another that confers resistance to hyperosmotic stress, and both pathways might be supporting tumorigenesis.Birt-Hogg-Dubé est un syndrome héréditaire qui augmente le risque de développer un cancer principalement des tumeurs et des kystes du rein. Folliculine (FLCN), hautement conservée à travers l'évolution, est la protéine suppresseur de tumeurs responsable de cette maladie. Malgré l'intense effort de recherche consacré depuis sa découverte, en 2001, la fonction de la protéine FLCN demeure obscure et comment son absence mène au cancer n'est pas encore défini. FLCN est un partenaire de la protéine-kinase (AMPK), un complexe impliqué dans la régulation du métabolisme cellulaire. Toutefois, les liens génétiques, fonctionnels, et phénotypiques n'ont pas été établis. Dans cette thèse, nous avons étudié le rôle de FLCN dans l'organisme modèle Caenorhabditis elegans (C. elegans) et dans des cellules de mammifères. Dans la première partie, nous démontrons que FLCN régule négativement l'AMPK, une fonction conservée à travers l'évolution. L'absence de la protéine FLCN active l'AMPK et induit l'autophagie, améliorant la bioénergétique cellulaire, et menant à une résistance avantageuse à plusieurs stress métaboliques incluant le stress oxydatif, l'anoxie, la chaleur et la carence en nutriments. Dans la deuxième partie, nous mettons en évidence la découverte d'une nouvelle fonction du complexe FLCN/AMPK dans la régulation du métabolisme du glycogène et dans l'adaptation au stress hyperosmotique chez C. elegans. Nous démontrons que l'absence de FLCN conduit à l'accumulation du glycogène et à la résistance aux conditions hyperosmotiques de manière dépendante de l'AMPK. Lors de l'exposition à un stress salin, les réserves de glycogène sont rapidement dégradées, conduisant à l'accumulation de l'osmolyte organique glycérol, un facteur essentiel pour l'adaptation de l'animal au stress hyperosmotique. Nous avons également prouvé que la régulation du métabolisme du glycogène par FLCN est conservée à travers l'évolution et que le glycogène s'accumule dans les reins de souris dépourvues de FLCN et dans la tumeur rénale d'un patient atteint par le syndrome BHD. Les résultats de cette partie démontrent un double rôle pour les réserves de glycogène: un réservoir d'énergie et un conservateur important de métabolites intermédiaires pour la production d'osmolytes en cas de besoin. Dans la troisième partie, nous étudions les changements transcriptionnels subis suite à l'absence de flcn-1 chez C. elegans. Les résultats de ce chapitre indiquent un chevauchement significatif avec des groupes de gènes induits par différents types de stress incluant le stress oxydatif, le stress hyperosmotique, et l'infection par des agents pathogènes. En outre, nous avons constaté que l'absence de flcn-1 chez C. elegans augmente la résistance aux pathogènes suggérant un rôle possible pour FLCN-1 dans la régulation de l'immunité innée. Nous avons aussi constaté que de nombreux gènes surexprimés chez les mutants nématodes flcn-1 sont régulés négativement chez les mutants hlh-30 et que la résistance au stress hyperosmotique des nématodes mutants flcn-1 est abolie par l'absence de l'hlh-30, soutenant un rôle possible de l'homologue de TFEB, HLH-30, dans la signalisation d'adaptation au stress en aval de la protéine FLCN-1. Ensemble, ces études ont démontré que FLCN régule négativement l'AMPK à travers l'évolution, et ont conduit à la découverte de deux voies distinctes de résistance au stress en aval de FLCN/AMPK, une voie de résistance aux stress métaboliques et une autre qui confère la résistance au stress hyperosmotique, et les deux voies pourraient soutenir la tumorigenèse

Randomized codon mutagenesis reveals that the HIV Rev arginine-rich motif is robust to substitutions and that double substitution of two critical residues alters specificity

The binding of the arginine-rich motif (ARM) of HIV Rev protein to its high-affinity site in stem IIB in the Rev response element (RRE) initiates assembly of a ribonucleoprotein complex that mediates the export of essential, incompletely spliced viral transcripts. Many biochemical, genetic, and structural studies of RevRRE IIB have been published, yet the roles of many peptide residues in Rev ARM are unconfirmed by mutagenesis. Rev aptamer I (RAI) is an optimized RRE IIB that binds Rev with higher affinity and for which mutational data are sparse. Randomized-codon libraries of Rev ARM were assayed for their ability to bind RRE IIB and RAI using a bacterial reporter system based on bacteriophage N-nut antitermination. Most Rev ARM residues tolerated substitutions without strong loss of binding to RRE IIB, and all except arginine 39 tolerated substitution without strong loss of binding to RAI. The pattern of critical Rev residues is not the same for RRE IIB and RAI, suggesting important differences between the interactions. The results support and aid the interpretation of existing structural models. Observed clinical variation is consistent with additional constraints on Rev mutation. By chance, we found double mutants of two highly critical residues, arginine 35 (to glycine) and asparagine 40 (to valine or lysine), that bind RRE IIB well, but not RAI. That an apparently distinct binding mode occurs with only two mutations highlights the ability of ARMs to evolve new recognition strategies and supports the application of neutral theories of evolution to proteinRNA recognition. Copyright (c) 2013 John Wiley & Sons, Ltd

The Transcription Factors TFEB and TFE3 Link the FLCN-AMPK Signaling Axis to Innate Immune Response and Pathogen Resistance

Summary: 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 confers pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, whereas ablation of total AMPK activity abolishes this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages is 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. : El-Houjeiri et al. show that loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression and phagocytosis in macrophages and confers pathogen resistance in C. elegans. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status to innate immunity. Keywords: TFEB, TFE3, FLCN, AMPK, innate immune response, pathogen resistance, autophagy, lysosomal biogenesis, phagocytosi

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival.

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally [...] which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function

Glycerol 3-phosphate phosphatase/PGPH-2 counters metabolic stress and promotes healthy aging via a glycogen sensing-AMPK-HLH-30-autophagy axis in C. elegans

Abstract Metabolic stress caused by excess nutrients accelerates aging. We recently demonstrated that the newly discovered enzyme glycerol-3-phosphate phosphatase (G3PP; gene Pgp), which operates an evolutionarily conserved glycerol shunt that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol, counters metabolic stress and promotes healthy aging in C. elegans. However, the mechanism whereby G3PP activation extends healthspan and lifespan, particularly under glucotoxicity, remained unknown. Here, we show that the overexpression of the C. elegans G3PP homolog, PGPH-2, decreases fat levels and mimics, in part, the beneficial effects of calorie restriction, particularly in glucotoxicity conditions, without reducing food intake. PGPH-2 overexpression depletes glycogen stores activating AMP-activate protein kinase, which leads to the HLH-30 nuclear translocation and activation of autophagy, promoting healthy aging. Transcriptomics reveal an HLH-30-dependent longevity and catabolic gene expression signature with PGPH-2 overexpression. Thus, G3PP overexpression activates three key longevity factors, AMPK, the TFEB homolog HLH-30, and autophagy, and may be an attractive target for age-related metabolic disorders linked to excess nutrients

Loss of <i>flcn-1</i> increases glycogen content, which mediates resistance to hyperosmotic stress.

<p>(A) Representative electron micrographs from longitudinal sections of the hypodermis in indicated nematodes strains exposed or not to 400mM NaCl for 16 hours. Arrows represent glycogen stores. Scale bars: 2μm. (B, C) Iodine staining (B) and quantification of staining intensities (C) of indicated worm strains treated or not with 400mM NaCl for 16 hours. Data represent mean ± SEM, n≥ 3. (D, E) Percent survival to 400mM NaCl of indicated worm strains treated with indicated RNAi. (F) Relative mRNA levels of indicated target genes in indicated strains with or without 400mM NaCl treatment for 2 hours. Data represent the mean ± SEM, n≥ 3.</p

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival

<div><p>Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor <i>FLCN</i>, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring <i>FLCN</i> mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using <i>Caenorhabditis elegans</i> and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.</p></div

Graphical representation of FLCN-1/AMPK hyperosmotic stress resistance pathway.

<p>Loss of <i>flcn-1</i> chronically activates AMPK and leads to glycogen accumulation under normal conditions. Upon exposure to hyperosmotic stress, glycogen is rapidly degraded leading to the production of glycerol and animal survival.</p