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'AMPactivated 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 VIII 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. LIST OF ABBREVIATIONS8-OHdG 8-hydroxy-2-deoxyguanosine ACC Acetyl-CoA carboxylases ADP Adenosine diphosphate AGE-1 Ageing alteration-1 AICAR 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside AMP Adenosine monophosphate AMPK 5'AMP-activated protein kinase APAF1 Apoptotic protease-activating factor-1 ATP Adenosine triphosphate C. elegans Caenorhabditis elegans.CAMKK Ca 2+ /calmodulin-activated protein kinase kinases CED-3 Cell death abnormal 3 CED-4 Cell death abnormal 4 CED-9 Cell death abnormal 9 CQ Chloroquine CREB cAMP-response element binding protein CYP 450 Cytochrome P450 DAF-16 Abnormal dauer formation DAF-2 Abnormal dauer formation 2 DAVID Database for Annotation, Visualization and Intergrated Discovery DENN Differentially expressed in normal cells and neoplasia dsRNA Double stransded RNA E. Coli Escherichia coli EGF Epidermal growth factor EM Electron microscopy ER stress Endoplasmic reticulum stress ERR Estrogen-related receptor FLCN Folliculin FNIP1 Folliculin-interacting protein 1 FNIP2 Folliculin-interacting protein 2 FNIPL FNIP-like FTC133 Follicular thyroid carcinoma cells 133 FUDR 5-fluoro-2'-deoxyuridine GABARAP GABA receptor-associated protein GEFs Guanine nucleotide exchange factors GLUT1 Glucose transporter 1 GLUT4 Glucose transporter 4 GPDH Glycerol-3-phosphate dehydrogenase GSY-1 Glycogen synthase 1 (C.elegans) GYS-1 Glycogen synthase 1 H 2 O 2 Hydrogen peroxide Chapter 4 For this chapter, I generated all figures and tables except panels 4.5D and 4.5E that were performed in the Irazoqui Lab.The microarray experiment was performed by Genome Quebec and results were analyzed by Greg Voisin.I validated the microarray results by qRT-PCR, and performed the gene ontology classification and gene overlap analysis.

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