Comprendre les fonctions moléculaires de l'ATPase EHD2 -­ un nouvel acteur de l'invasion des cancers du sein

par Satish Kailasam Mani

Projet de thèse en Aspects moléculaires et cellulaires de la biologie

Sous la direction de Christophe Lamaze.

Thèses en préparation à Paris Saclay , dans le cadre de École doctorale Signalisations et réseaux intégratifs en biologie (Kremlin-Bicêtre, Val-de-Marne) , en partenariat avec Chimie biologique des membranes et ciblage thérapeutique (Paris, Institut Curie) (laboratoire) et de Université Paris-Sud (établissement de préparation de la thèse) depuis le 01-12-2016 .


  • Résumé

    Les cavéoles, petites invaginations de la membrane plasmique (50-100 nm), sont composées principalement de cavéolines (Cav1/Cav2/Cav3). Bien que les cavéoles et la cavéoline 1 (Cav1) aient été impliquées dans des processus de croissance tumorale et de métastase, leur fonction exacte dans les cancers reste mal comprise. Eps15 homology domain-containing protein 2 (EHD2) est une autre protéine récemment décrite associée aux cavéoles. La très faible expression d'EHD2 dans des gliomes et des carcinomes épidermoïdes oesophagiens (ESCC) suggère son importance dans le développement et la progression des cancers. Cependant le mécanisme moléculaire expliquant le lien entre EHD2 et cancer ou métastase n'est toujours pas clair. Mon laboratoire d'accueil a mis en évidence pour la première fois le rôle des cavéoles dans la mécano-transduction des cellules cancéreuses et des résultats non publiés ont identifié EHD2 comme une protéine clef présentant des propriétés de « mécano-récepteur » et de mécano-transduction. De plus EHD2 est ressorti comme candidat dans un crible protéomique des Rab GTPases, des régulateurs du trafic intracellulaire (laboratoire du Dr. B. Goud, UMR144, IC). Pendant mon stage de M2 dans le laboratoire, j'ai pu confirmer par des techniques de double hybride (levure) et de co-immunoprécipitation l'interaction d'EHD2 avec certaines Rabs spécifiques connues pour réguler les endosomes de recyclage et le réseau trans-golgien (TGN). J'ai aussi confirmé que la déplétion d'EHD2 dans les cellules épithéliales de rétine humaines (RPE-1) cause une fragmentation de l'appareil de Golgi. L'imagerie en temps réel montre une diminution significative de la population de vésicules de transport sortant du Golgi dans ces mêmes conditions. Ces résultats préliminaires suggèrent qu'EHD2 jouerai un rôle dans le trafic membranaire depuis le TGN. Etant donnés ces premiers résultats, je vais dans un premier temps identifier et caractériser de nouveaux partenaires d'EHD2 et étudier le profil d'expression génétique de lignées cellulaires épithéliales de cancer du sein en condition d'étirement ou au repos. De plus, EHD2 est exporté dans le noyau en condition de stress mécanique et y régule la transcription de gènes spécifiques. Il sera donc intéressant de comparer les profils de transcription de ces gènes dans des lignées de cellules exprimant des quantités différentes d'EHD2. J'étudierai aussi le rôle d'EHD2 dans des voies de signalisation en réponse à des stress mécanique grâce à la plateforme de Reverse Phase Protein Assay (RPPA) de l'Institut Curie. En parallèle, j'essayerai de comprendre le rôle d'EHD2 et de son association au Golgi et aux endosomes de recyclage ainsi que de caractériser la dynamique moléculaire de Cav1 et EHD2 dans des cellules cancéreuses soumises ou non à un stress mécanique. En conclusion, nous cherchons à apporter une compréhension du mécanisme d'action d'EHD2 dans l'agressivité des cancers du sein et à identifier des nouveaux régulateurs. Ils seront autant de cibles potentielles qui pourraient permettre une thérapie ciblée des cancers du sein triple-négatif.

  • Titre traduit

    Analyzing the molecular functions of the ATPase EHD2, a new actor in invasive breast cancers


  • Résumé

    Summary (max. 8000 characters) Caveolae are small (50-100 nm) invaginations in the plasma membrane, rich in sphingolipids and cholesterol, and composed mainly of caveolins (Cav1/Cav2/Cav3) as the structural components. Cells subjected to repeated mechanical stress, such as adipocytes, endothelial and muscle cells, are highly enriched in caveolae. Although caveolae and/or caveolin-1 (Cav1) have long been involved in processes associated with tumor growth and metastasis, the function of caveolae in cancer remains poorly understood. Another protein recently established to be associated with caveolae is the Eps15 homology domain-containing protein 2 (EHD2). In the recent past, EHD2 has been implicated as a tumor suppressor gene in gliomas and esophageal squamous cell carcinoma. These studies have brought attention to the role of EHD2 in the development and progression of cancers. However, molecular data on the relationship between EHD2 and cancer and the role of EHD2 in metastasis is unclear. Biomechanical factors and their possible alteration in the tumor mass have recently emerged as crucial factors in the control of tumor evolution. In this context, my host laboratory established a new role for caveolae in mechanosensing wherein caveolae rapidly flatten out in response to mechanical stress thereby providing additional membrane to buffer the resultant increase in membrane tension and maintain homeostasis. The flattening of caveolae also leads to rapid disassembly and release of the caveolar coat proteins including Cav1 and cavins. My host laboratory hypothesized that the mechanical release of caveolae constituents could mediate mechanotransduction events. In this regard, my host laboratory has established a previously unsuspected role for EHD2 in caveolae mechanosensing and mechanotransduction and showed that this new function was altered in Triple Negative Breast Cancers (TNBCs). We found that when cells were subjected to mechanical stress (either by hypo-osmotic shock or by uniaxial stretching), EHD2 is released from the neck of caveolae at the plasma membrane, sumoylated, and translocated to the nucleus where it inhibits the transcription of caveolae-related genes (Cav1, Cav2, Cavin1 and Cavin2). Moreover, it was also observed that EHD2 is a negative regulator of cell migration and invasion, and that low gene expression was in-turn associated with an invasive profile in a cohort of 500 breast cancer patients. These results show for the first time the role of caveolae in cancer mechanotransduction and identify EHD2 as a key protein with both mechanosensing and mechanotransducing properties. In addition, proteomics data of Rab GTPases (from Dr. B.Goud's laboratory, UMR 144, Institut Curie) indicated a hit for EHD2 with the small GTPases Rab6A/A' and Rab8A, key regulators of intracellular transport within the Golgi apparatus and the recycling endosomes. During my Master 2 project in the laboratory of Dr. Christophe Lamaze (UMR 3666, Institut Curie), I confirmed these interactions through Co-immunoprecipitation and yeast two-hybrid assays and found that EHD2 interacts with Rab6A/A' and Rab11A but not with Rab8A. This suggested a role for EHD2 involving the recycling endosomes and the Trans-Golgi network (TGN). I also confirmed that knockdown of EHD2 in Retinal Pigmentary Epithelial-1 (RPE-1) cells resulted in fragmentation of the Golgi and live-cell imaging indicated a significantly reduced population of transport vesicles exiting the Golgi apparatus in siEHD2 RPE-1 cells. These preliminary results suggest that EHD2 plays a role in the integrity of the Golgi apparatus and lead us to hypothesize a new role for EHD2 in membrane trafficking possibly from the TGN. In view of these preliminary results, my research objectives are: 1. To Identify and characterize novel interaction partners of EHD2 and perform gene expression profiling in breast epithelial cell lines under resting and stretching conditions: For this, GFP-Trap pull down experiments in EHD2-GFP transfected TNBC (Hs578T and MDA-MB-436) and normal breast epithelial cell lines (MCF10-2A) will be performed under normal and stretching conditions. The GFP-Trap immunoprecipitants from these cells will be analyzed by the mass spectrometry core facility of Institut Curie for identifying novel EHD2 interaction partners. Since EHD2 translocates to the nucleus under mechanical stress and regulates gene transcription, we expect to reveal a difference in gene expression profiling between the cell lines that express varying levels of EHD2. Microarray analysis/DNA chip analysis will also be performed in these cell lines under resting and stretching conditions in order to determine the difference in gene expression profiles and identify genes regulated by EHD2. 2. To identify signaling pathways affected by EHD2 under mechanical stress: The Reverse Phase Protein Array (RPPA) platform at Institut Curie will be used for this purpose. The RPPA platform allows high throughput screening of proteins that are involved in signal transduction. The principle of this technology is that protein activation/inactivation state can be recognized by specific antibodies (more than 500 available) to detect modified proteins (e.g. phosphorylation). Classical cell biology and imaging methods will be used to validate the RPPA candidates. I will also evaluate their impact of EHD2 mediated mechanosensing and mechanotransduction by applying drugs, siRNA and mutants to interfere with identified pathways. These data will be interpreted in correlation with the gene candidates and proteomics hits identified in 1. 3. To elucidate the role of EHD2 and its association with the Golgi apparatus and recycling endosomes: The molecular mechanisms that regulate the trafficking of Cav1 and EHD2 from the plasma membrane to the nucleus and back to the plasma membrane (after stress release) to reassemble functional plasma membrane caveolae are unclear. My Master 2 project preliminary data indicates an association of EHD2 with Rab6A/A' and Rab11A, suggesting that EHD2 trafficking may also take place between the TGN and recycling endosomes. Hence, we would like to gain mechanistic insights and further dissect the role of EHD2 and its association with Golgi and recycling endosomes, in light of it's shuffling between the plasma membrane and the nucleus. Using high resolution imaging (TIRF, FLIM-FRET, FRAP, confocal, spinning disk) available at the Nikon Imaging Centre of the IC and various assays (endocytosis assays, electron microscopy etc.) /molecular tools (fluorescently-tagged EHD2, Cav1 etc.), I will characterize the molecular dynamics of Cav1 and EHD2 under resting and stretching conditions in normal and cancer breast cells. In conclusion, with the help of customized stretching devices and high throughput screening (Proteomics, DNA chip/microarray analysis, RPPA) combined with our preliminary data, we seek to provide mechanistic insights into the role of EHD2 in breast cancer aggressiveness and identify key new regulators that will represent potential new therapeutic targets that are needed to treat triple negative breast cancers.