Dissertations / Theses on the topic 'HIPPO-PATHWAY-YAP'

Yes-associated protein (YAP) is a co-transcription factor that acts downstream of the evolutionarily conserved Hippo pathway. Canonically, this pathway regulates tissue growth in flies and mammals, by controlling the nuclear localization of YAP. Interestingly, in addition to the conserved functions of this pathway, some of the mammalian orthologs of pathway components (e.g. MST, RASSF1, WW45, and LATS) have been shown to localize to the nucleus and alterations in their expression induces alterations in mitotic processes, suggesting additional roles for these proteins in mitosis. In this thesis, I have uncovered a role for the Hippo pathway effector protein, YAP, in cytokinesis. YAP was found to localize to the central spindle and cytokinetic midbody and biochemical analysis demonstrated that YAP is phosphorylated by the mitotic regulatory kinase CDK1 during mitosis. Time-lapse microscopy of cells in which YAP was downregulated by shRNA revealed that reduction in YAP expression causes a delay in abscission and induces a cytokinesis phenotype associated with increased contractile force, membrane blebbing and bulges, and abnormal spindle orientation; consequently, this leads to an increased frequency of multinucleation, micronuclei, and aneuploidy. Expression of or expression of a variant of YAP that could not be phosphorylated at the mitotic phosphoacceptor sites induced a phenotype similar to that of YAP knockdown, suggesting that mitotic YAP phosphorylation is critical for YAP’s function in cytokinesis. Reduction in YAP expression also disrupted the localization of ECT2, MgcRacGap, Anillin, and RHOA, proteins important for cleavage furrow function during cytokinesis, Reduction of YAP also increased levels of phosphorylated myosin light chain, which activates myosin II contractile activity. These findings suggest that YAP is required for proper coordination of these contractile processes involved in cytokinesis. In addition, the YAP mitotic phosphorylation sites are required for interaction with the scaffold polarity protein PATJ, and PATJ co-localizes with YAP at the cytokinesis midbody. PATJ knockdown induces cytokinesis defects and spindle orientation alterations similar to those detected in YAP- depleted cells or cells expressing a non-phosphorylatable mutant of YAP. This study reveals an unanticipated role for YAP during mitosis and implicates YAP in processes that control the proper organization of cytokinesis machinery through interaction with the polarity protein PATJ. Thus, these studies demonstrate a previously unanticipated role for YAP that is independent of its activity as a transcriptional coactivator. In addition, although YAP is known to function as a potent oncogene, our findings indicate that YAP may also act as a tumor suppressor in certain contexts since loss of YAP could lead to genetic alterations associated with defective cytokinesis. These studies add to the complexity of YAP regulation in cancer as well as in normal development and provide a framework for future studies in a new area of Hippo pathway biology.
Medical Sciences

The mechanism of body and organ size control is an unsolved puzzle. Initially characterized in Drosophila melanogaster, the Salvador/Warts/Hippo (Hippo) signalling pathway, highly conserved throughout evolution, defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis, and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. Previously Willin/FRMD6 has been proposed as the human orthologue of Expanded and, to date, little is known about the functional role of Willin in mammalian cells. My study elucidated the mechanism by which Willin antagonizes the transcriptional co-activator YAP. In MCF10A cells, Willin ectopic expression antagonizes YAP-induced epithelial-mesenchymal phenotypes via YAP Ser127 phosphorylation site. Loss of Willin expression attenuates MST1/2, LATS1, and YAP phosphorylation promoting YAP's oncogenic transformation activity in vitro, as analysed by its ability to display epithelial-to-mesenchymal transition (EMT) features. These biological outputs are YAP dependent. These data support the involvement of Willin in the regulation of the mammalian Hippo signalling activity by activating the core Hippo pathway kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but the functional consequences of these biochemical changes have not been previously addressed. I showed that in MCF10A cells, loss of KIBRA expression displays EMT features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the Ser 127 phosphorylation site and I showed that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Willin/FRMD6 was first identified in rat sciatic nerve, which is composed of Schwann cells and fibroblasts. To elucidate the function of Willin in the mammalian sciatic nerve, I showed that Willin is predominantly expressed in fibroblasts and that its expression activates the Hippo signalling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These evidence show that Willin modulates sciatic nerve fibroblast activity, indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.

The Hippo signalling pathway is an evolutionarily conserved kinase cascade responsible for the cell proliferation, tissue growth and apoptosis during development and its dysregulation contributes to tumourigenesis. This signalling pathway was initially discovered in Drosophila and soon after that, it was shown to be highly conserved in mammals. The core Lats kinases of this tumour suppressive pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Latest reports revealed various E3 ubiquitin ligases to negatively regulate the Hippo pathway through ubiquitination, yet few deubiquitinating enzymes have been described. In the present study, we report USP9X deubiquitinating enzyme as an essential regulator of the central components of this pathway. USP9X interacted strongly with Lats2 kinase and to a lesser extent with WW45, Kibra and Angiomotin family proteins. The knockdown of USP9X resulted in notable downregulation and destabilisation of Lats kinase and to lesser extents WW45, Kibra and Amot. This resulted in enhanced nuclear localisation of YAP and TAZ accompanied with activation of their target genes, CTGF and CYR61. USP9X was shown to stabilise Hippo components through its deubiquitinating activity. USP9X enzyme defective mutant lost the activity to stabilise Lats2, WW45, Kibra and Angiomotins through deubiquitination, leading to their ubiquitination. In the absence of USP9X, cells exhibited epithelial to mesenchymal transition phenotype and additionally gained anchorage-independent growth in soft agar. Moreover, USP9X knockdown disrupted acinar organisation of breast cells in three-dimensional acini cultures. In addition, YAP/TAZ target gene activation in USP9X knockdown cells could be rescued by knockdown of YAP, TAZ and TEAD2. Lastly, USP9X protein expression showed a positive correlation with Lats kinases, but negative correlation with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. The results strongly indicate that USP9X cooperates with Lats2 and other important Hippo components to suppress tumour growth.

One of the key questions in developmental biology is how tissue growth is controlled to give rise to organs of specific sizes and shapes. Although some genes and pathways involved in the genetic and environmental control of tissue growth have been uncovered, the understanding of this process remains incomplete. In order to find new regulators of growth we carried out an in vivo RNAi screen in the Drosophila wing. I participated in the validation of candidate genes from the screen and identified the mask gene as an essential regulator of tissue growth acting in the Hippo signaling pathway. This pathway acts via the Yorkie (Yki)/Yes-associated protein (YAP) transcriptional co-activator to control tissue growth in both Drosophila and mammals. Yki/YAP translocates from the cytoplasm to the nucleus to activate target genes, a process that is negatively regulated by the Warts kinase, one of the core components of the Hippo pathway. I found that Mask is an essential positive regulator of Yki acting downstream of Warts. Mask is required for normal tissue growth, for the expression of Yki target genes and for the overgrowth phenotype caused by Yki overexpression. Mask binds to Yki and the two proteins translocate from the cytoplasm to the nucleus together in response to various stimuli. My results show that Mask acts in the nucleus to promote Yki target gene activation. Finally, Mask’s function appears to be conserved in humans, as two human homologues of Mask (hMask1 and hMask2) translocate with YAP to the cytoplasm upon cell contact inhibition, and we demonstrate that one of these homologues promotes YAP’s transactivation function.

YAP and TAZ, by orchestrating cell proliferation, cell death and cell-fate decisions, are key players of a complex network of signaling pathways acting during development. Deregulation of YAP/TAZ signaling causes robust organ overgrowth during organogenesis, which translates to loss of tissue homeostasis in the adult and consequent cancer development. YAP/TAZ are transcriptional co-activators that interact with TEAD transcription factors to promote cell proliferation and survival. Their transcriptional activity is regulated by nucleocytoplasmic shuttling and nuclear accumulation, which are controlled by the Hippo kinase cascade, but also by mechanical cues sensed by the cell and by other pathways. Among these, Wnt/β-catenin takes on a particular relevance, since it was recently shown to regulate YAP/TAZ activity through AXIN-mediated sequestration of YAP/TAZ in the β-catenin destruction complex. Here, we describe the generation, validation and characterization of a novel biosensor zebrafish reporting the activity of Yap/Taz. It expresses nuclear mCherry, eGFP or the destabilized green fluorescent protein VenusPEST under the control of a promoter fragment of the human YAP/TAZ target gene CTGF, that contains 3 TEAD DNA-binding sites. Several independent founder fish transmitting the transgene to the germline were identified and used to establish the stable reporter lines. All stable transgenic fish shared a similar expression pattern, which was maintained in subsequent generations. Knockdown and overexpression approaches were used to validate the reporter. Co-injection of two morpholinos targeting Yap and Taz pre-mRNAs reduced the reporter signal, whereas injection of mRNAs coding for a constitutively active form of Yap, Taz and Tead (YAP-5SA, TAZ-4SA, TEAD-VP16) increased it. The CTGF-based transgenic lines represent therefore bona fide Yap/Taz reporters. During development, strong reporter signal is visible mainly in the lens and otic vesicles, the pharyngeal arches, the heart, the pectoral fin and the vasculature, but the reporter protein expression is also detected in many other tissues and organs. The almost ubiquitous activation of Yap/Taz observed during early embryogenesis, consistent with the general role of YAP/TAZ in promoting cell proliferation and organ growth, is largely silenced in the adult fish, where the reporter signal is restricted to the lens, the ovary, the heart and the whole vasculature. We also showed that the CTGF-based biosensor zebrafish is able to report Yap/Taz activation during larval and adult fin regeneration, as expected from the role that YAP/TAZ signaling plays in the regenerative processes. The zebrafish CTGF-based reporter permitted to show in a living organism during development the regulation that the Wnt/β-catenin pathway exerts on Yap/Taz activity. Our results in terms of variations of the reporter signal, after both genetic and pharmacological modulation of the Wnt pathway activity, are in accordance with the model recently depicted in vitro. The general and sustained reporter activity we observed in the endothelium during embryogenesis suggested a functional involvement of Yap/Taz signaling in developmental angiogenesis. Yap/Taz knockdown impaired the intersegmental vessels (ISVs) growth, while the overactivation of Yap/Taz-mediated transcription caused an aberrant sprouting from the ISVs. The vessel sprouting-promoting capacity of Yap/Taz is cell-autonomous, as the same phenomenon was observed by expressing TAZ-4SA under the control of an endothelium-specific promoter. The CTGF-based zebrafish reporter is a new powerful tool to study in vivo Yap/Taz pathway activation, with possible applications in drug screening, regeneration and cancer biology. It permitted to confirm in vivo during development the crosstalk between Wnt/β-catenin and Yap/Taz pathways and to discover a novel role of Yap/Taz in vessel sprouting, suggesting a pro-angiogenic function of YAP/TAZ transcriptional activity.
YAP e TAZ, orchestrando la proliferazione, la morte e il differenziamento cellulari, rappresentano elementi chiave di una complessa rete di vie di segnalazione che agiscono durante lo sviluppo. L’alterazione della segnalazione YAP/TAZ causa una crescita fuori controllo degli organi durante l’organogenesi, che si traduce nella perdita dell’omeostasi tissutale nell’adulto e conseguente sviluppo tumorale. YAP/TAZ sono co-attivatori trascrizionali che interagiscono con i fattori di trascrizione TEAD per promuovere la proliferazione e la sopravvivenza cellulari. La loro attività trascrizionale è regolata dal trasporto nucleo-citoplasmatico e dall’accumulo nucleare, che sono controllati dalla cascata chinasica della via di Hippo, ma anche dagli stimoli meccanici percepiti dalla cellula e da altre vie. Fra queste, la via di Wnt/β-catenina assume una particolare rilevanza, dal momento che è stato recentemente dimostrato che essa regola l’attività di YAP/TAZ attraverso il loro sequestro nel complesso di degradazione della β-catenina mediato da AXIN. In questa tesi vengono descritte la generazione, la validazione e la caratterizzazione di un nuovo zebrafish biosensore che riporta l’attività di Yap/Taz. Esso esprime le proteine mCherry nucleare, eGFP o la proteina verde fluorescente destabilizzata VenusPEST sotto il controllo di un frammento promotoriale del gene umano CTGF target di YAP/TAZ, contenente 3 siti di legame per TEAD. Sono stati identificati diversi pesci fondatori indipendenti in grado di trasmettere il transgene alla linea germinale, i quali sono stati utilizzati per instaurare le linee reporter stabili. Tutti i pesci transgenici condividevano un pattern di espressione similare, mantenuto nelle generazioni successive. Per validare il reporter sono stati usati approcci di downregolazione e overespressione. La co-iniezione di due morfolini diretti contro i pre-mRNA di Yap e Taz ha ridotto il segnale reporter, mentre l’iniezione di mRNA codificanti per una forma costitutivamente attiva di Yap, Taz o Tead (YAP-5SA, TAZ-4SA, TEAD-VP16) lo ha aumentato. Le linee transgeniche basate sul gene CTGF rappresentano perciò bona fide dei reporter dell’attività di Yap/Taz. Durante lo sviluppo, un forte segnale reporter è visibile principalmente nella lente, la vescicola otica, gli archi faringei, il cuore, la pinna pettorale e la rete vascolare, ma l’espressione della proteina reporter è rilevabile in molti altri tessuti e organi. L’attivazione quasi ubiquitaria di Yap/Taz osservata durante l’embriogenesi precoce, consistente con il ruolo generale di YAP/TAZ nel promuovere la proliferazione cellulare e la crescita degli organi, è ampiamente silenziata nel pesce adulto, dove il segnale reporter è ristretto a lente, ovario, cuore e intera rete vascolare. Lo zebrafish biosensore è anche in grado di riportare l’attivazione di Yap/Taz durante la rigenerazione della coda nella larva e nell’adulto, come atteso dal ruolo che riveste la segnalazione YAP/TAZ nei processi rigenerativi. Lo zebrafish reporter basato sul gene CTGF ha permesso di mostrare in un organismo vivente durante lo sviluppo la regolazione che la via di Wnt/β-catenina esercita sull’attività di Yap/Taz. I nostri risultati in termini di variazione del segnale reporter, in seguito alla modulazione genetica e farmacologica dell’attività della via di Wnt, sono in linea con il modello disegnato di recente in vitro. L’attività generale e sostenuta del reporter nell’endotelio durante l’embriogenesi ha suggerito un coinvolgimento funzionale della segnalazione Yap/Taz nell’angiogenesi precoce. La downregolazione di Yap/Taz è risultata in una compromissione della crescita dei vasi intersegmentali (ISVs), mentre l’attivazione spinta della trascrizione mediata da Yap/Taz ha causato un ramificarsi anomalo degli ISVs. La capacità di Yap/Taz di promuovere tale ramificazione vascolare è “cell-autonomous”, dal momento che lo stesso fenomeno è stato osservato esprimendo TAZ-4SA sotto il controllo di un promotore endotelio-specifico. Lo zebrafish reporter sviluppato è un nuovo potente strumento per studiare in vivo l’attivazione della via di Yap/Taz, con possibili applicazioni nello screening farmacologico e nella biologia della rigenerazione e del cancro. Ha permesso di confermare in vivo durante lo sviluppo l’interazione fra le vie di Wnt/β-catenina e Yap/Taz e di scoprire un nuovo ruolo di Yap/Taz nella ramificazione vascolare, suggerendo una funzione pro-angiogenica dell’attività trascrizionale di YAP/TAZ.

Une cellule souche est capable de s’auto-renouveler et de générer des cellules différenciées après division cellulaire. La duplication complète de son génome doit être exempte d'erreurs afin d'éviter la propagation aux cellules filles de mutations délétères. Chez les eucaryotes, il a été montré que des segments d’ADN sur les chromosomes se répliquent de manière coordonnée et à des moments définis pendant la phase de synthèse, un processus appelé programme spatio-temporel de réplication de l'ADN (RT). Des changements majeurs dans le RT sont corrélés avec les changements de détermination des cellules souches et associés à l'organisation et à l’expressivité du génome. Malgré ce rôle central, les mécanismes qui sous-tendent le contrôle du RT restent méconnus. Mon laboratoire a mis en évidence que YAP, l'effecteur en aval de la voie de signalisation Hippo impliquée dans la croissance cellulaire, régule la vitesse et la chorégraphie de la réplication de l’ADN des cellules souches rétiniennes chez l’amphibien xénope. Ces données révèlent YAP comme un nouvel acteur moléculaire dans le contrôle du RT. Pour tester l’implication directe de YAP dans la dynamique de réplication de l’ADN, nous avons tiré profit du système in vitro d’extraits d'œufs de xénope dans lequel toutes les étapes du processus sont reproduites de manière synchrone. Nous montrons que YAP est recruté à la chromatine pendant la réplication et que ce processus se produit seulement après la phase d’initiation des origines de réplication. Des extraits déplétés de la protéine YAP présentent une accélération de la vitesse de réplication et une augmentation du nombre de sites d’activation de la synthèse de l’ADN. Par ailleurs, nous avons identifié RIF1 (Rap1-Interacting Factor 1 ou Replication Timing Regulatory Factor 1), un des rares régulateurs connus du RT, comme un nouveau partenaire de YAP. Comme pour YAP, la perte de fonction de RIF1 dans les embryons de xénope conduit à un phénotype de petit œil et à la dérégulation du RT dans les cellules souches rétiniennes.Dans l'ensemble, nos résultats montrent l’implication de YAP dans le contrôle de la dynamique de réplication de l’ADN et révèlent RIF1 comme un nouveau partenaire dans ce processus. Ce travail ouvre de nouvelles perspectives d’étude quant à l’importance biologique de cette interaction YAP-RIF1 dans le contrôle du RT et sa pertinence comme cible pour influencer le devenir des cellules souches
Stemness could be defined as a state in which a cell is able to self-renew and/or to differentiate after cell division. Before this happens, exhaustive duplication of the genome free of errors must occur in order to avoid deleterious mutations, a hallmark of cancer. Thus, DNA replication is particularly important to stem cells because of their continuous division capacities. Regarding DNA replication in eukaryotes, it was discovered that segments of chromosomes close in space, replicate in a coordinated manner during S phase, a process called replication timing. Moreover, major changes in replication timing correlate with cell differentiation, 3D chromatin architecture and transcription. However, the molecules that govern its regulation are poorly understood. Previously, my laboratory found that YAP, the downstream effector of the Hippo pathway, regulates S phase progression of retinal stem cells in Xenopus laevis. To test YAP function in the direct control of replication timing, we took advantage of the powerful in vitro DNA replication system of X. laevis egg extracts. Briefly, we discovered that YAP is recruited to replicating chromatin dependently of origin licensing. In addition, YAP depleted extracts showed increased DNA synthesis and origin activation; revealing that YAP normal function is to slow-down replication by limiting origin firing. Interestingly, we found Rif1, a major regulator of replication timing, as a novel partner of YAP. In vivo, Rif1 expression overlaps that of Yap within the stem cell compartment of the Xenopus retina. Knockdown of Rif1 leaded to a small-eye phenotype and alterations in replication foci of retinal stem cells, resembling the effect observed in YAP deficient cells. Finally, early-embryonic depletion of both molecules resulted in a strikingly acceleration of cell division.Altogether, our findings unveil YAP implication in the regulation of replication dynamis and show Rif1 as a novel partner. Further investigation to analyze this interaction would help us to understand the biological relevance in the control of replication timing and whether it could be used as a target in regenerative medicine

The paralogous multi-functional adaptor proteins YAP and TAZ are nuclear effectors of the Hippo pathway, a central regulator of developmental organ size control, tissue homeostasis and tumour suppression. YAP/TAZ target the TEAD transcription factor family to promote cell survival and inhibit apoptosis. TEAD proteins contain a DNAbinding domain and a YAP/TAZ interaction domain. PCR analysis of medaka fish TEAD cDNA revealed the presence of alternative TEAD splice-forms with variations at the C-terminus of the DNA-binding domain. Structural analysis indicated the YAPbinding domain of TEAD proteins is folded and globular. NMR spectroscopy showed that the TEAD binding domain of YAP does not contain secondary structure. YAP and TAZ both contain WW domains, which are small protein-protein interaction modules. Two YAP isoforms are known, YAP1 and YAP2 that contain one and two WW domains, respectively. To date, only a single WW isoform of TAZ has been described. PCR analysis of medaka TAZ cDNA identified both single WW and tandem WW isoforms of TAZ. NMR spectroscopy was used to characterise structural, conformational, and peptide binding features of the tandem WW domains from YAP and TAZ. The YAP WW2 solution structure confirms that the domain has the canonical anti-parallel β-sheet WW fold. WW1 of YAP and both WW domains of TAZ undergo conformational exchange. The region linking the two WW domains is flexible and allows interaction of both WW domains with peptides containing single and dual PPxY binding motifs. In addition to YAP and TAZ, tandem WW domains are also present in the core and upstream Hippo pathway proteins Salvador and Kibra. Both proteins contain one atypical WW domain; the tandem WW domains of these two proteins are unstable. Understanding structure and function of Hippo pathway components could contribute to drug development and will also contribute to knowledge of protein folding and interactions.

Les maladies dégénératives de la rétine sont une des causes principales de cécité. Parmi les différentes stratégies thérapeutiques actuellement étudiées, notre équipe s’intéresse au potentiel régénératif de la rétine. Une source cellulaire d'intérêt sont les cellules de Müller, principal type de cellules gliales de la rétine capables de se réactiver en cas de dégénérescence, un processus appelé gliose réactive, et dans certaines espèces d’adopter des caractéristiques de cellules souches. Si un tel processus confère la capacité de régénérer la rétine chez les téléostéens, il est cependant largement inefficace chez les mammifères. Avoir une meilleure connaissance des mécanismes moléculaires sous-jacents pourrait aider à transformer leur potentiel de régénération en nouvelles stratégies thérapeutiques en condition pathologique de dégénérescence rétinienne. Dans ce contexte, mon laboratoire s'est focalisé sur l'effecteur terminal de la voie Hippo, le cofacteur de transcription YAP, dont il a été démontré qu'il stimule la régénération de plusieurs organes en cas de lésion. Dans la rétine, YAP est spécifiquement exprimé dans les cellules de Müller et son niveau d’expression augmente en cas de lésion. Cependant, sa fonction dans l'homéostasie rétinienne, et en particulier son rôle dans la régénération rétinienne, sont encore inconnus. La première partie de ma thèse visait donc à décrypter la fonction de YAP dans les cellules de Müller de souris dans des conditions physiologiques et pathologiques. Nous avons révélé que YAP joue un rôle central dans l'homéostasie des cellules de Müller et en tant que tel, est un acteur clé de la survie des cônes au cours du vieillissement. En cas de lésion rétinienne, nous avons montré que YAP est essentiel pour la réactivation des gènes du cycle cellulaire qui accompagne normalement la gliose réactive. Dans ce contexte, nous avons également trouvé une interaction fonctionnelle entre YAP et la voie de signalisation EGFR, suggérant une fonction de YAP en tant qu’intégrateur des réseaux de signalisation mis en jeu dans le contexte régénératif. J'ai également constaté que la suractivation de YAP est suffisante pour induire la reprogrammation des cellules de Müller de souris en cellules hautement prolifératives […]. Dans l'ensemble, ce travail met en évidence le rôle critique de YAP dans la sortie de quiescence des cellules de Müller chez les mammifères et révèle ainsi une cible potentielle pour la médecine régénérative. La deuxième partie de mon projet de doctorat naît des découvertes émergentes mettant en évidence les voies inflammatoires comme régulateurs du processus de régénération. […] De plus, des découvertes récentes sur le rôle de YAP dans la régulation du processus inflammatoire m’ont conduit à faire l'hypothèse qu'il pourrait jouer un rôle dans la relation entre l'inflammation et la régénération rétinienne. J'ai donc cherché à étudier le rôle joué par l'inflammation sur le comportement des cellules de Müller de souris, et à comprendre comment YAP s'inscrit dans cette interaction. J'ai découvert de manière inattendue qu'un contexte pro-inflammatoire établi par les cellules microgliales stimule la prolifération des cellules de Müller de souris dans des explants rétiniens. De plus, mes résultats ont montré que cet effet mitogène se produit de manière dépendante de YAP. Par ailleurs, j'ai découvert que l'effet de la surexpression de YAP sur la prolifération des cellules de Müller peut être potentialisé par un environnement pro-inflammatoire et aboli en cas d’ablation des microglies. Enfin, nous avons constaté que, à son tour, YAP régule des cytokines inflammatoires clés. Dans l'ensemble, cette partie de mon projet permet non seulement d’approfondir nos connaissances concernant l'impact de l'inflammation sur le comportement des cellules de Müller de la souris, mais met également en évidence YAP en tant qu'acteur clé dans la connexion entre l'inflammation et la régénération rétinienne
Degenerative diseases of the retina are one of the main causes of blindness. Among the various therapeutic strategies currently being studied, our team is focusing on the regenerative potential of the retina. One cellular source of interest are Müller cells, the main type of glial cells in the retina capable of reactivating in case of degeneration, a process called reactive gliosis, and in some species adopting certain characteristics of stem cells. If such a process sustains powerful regeneration abilities in teleosts, it is however largely inefficient in mammals. Hence, increasing our knowledge of the molecular mechanisms underlying the behaviour of these cells under pathological conditions may help turning their regenerative properties into new therapeutic strategies. In this context, my laboratory focused on the terminal effector of the Hippo pathway, the co-transcriptional factor YAP, which has been shown to stimulate regeneration of several injured organs. In the retina, YAP is specifically expressed in Müller cells and upregulated in case of damage. However, its function in retinal homeostasis, and its role in retinal regeneration remained unknown.The first part of my PhD aimed at deciphering YAP function in mouse Müller cells in both physiological and pathological conditions. In essence, we revealed a central role of YAP in Müller cell-dependent retinal homeostasis and as such, as a key player for cone survival during aging. In case of retinal damage, we showed that YAP upregulation is critical for cell-cycle gene reactivation that normally accompanies reactive gliosis. In this context, we also found a functional interaction between YAP and the EGFR signaling pathway, supporting a function of YAP as a hub within the complex signaling network of key regenerative signaling pathways. I also found that YAP overactivation is sufficient to induce mouse Müller cell reprogramming into highly proliferative cells, mimicking a fish or amphibian condition, when Müller cells spontaneously proliferate upon injury. As a whole, this work highlights the critical role of YAP in driving mammalian Müller cells to exit quiescence and thus reveals a potential target for regenerative medicine.The second part of my PhD project stemmed from the emerging discoveries highlighting inflammatory pathways as regulators of the regenerative process. Although inflammation is considered to hamper retinal regeneration in mammals, there are no studies regarding the influence of inflammation on mouse Müller cell-dependent regenerative process. In addition, recent discoveries on the role of YAP in the regulation of the inflammatory process lead to the hypothesis that it could play a role in the relationship between inflammation and retinal regeneration. I thus aimed at investigating the role played by the injury-induced inflammation on mouse Müller cell behavior and how YAP fits in this interplay. I unexpectedly discovered that a microglial-dependent pro-inflammatory context stimulates mouse Müller cell proliferation in retinal explants. Importantly, my results showed that this mitogenic effect occurs in a YAP-dependent manner. Moreover, I uncovered that the effect of YAP overexpression on Müller cell proliferation can be potentiated by a pro-inflammatory environment, and abolished upon microglia depletion. Finally, we found that, in turn, YAP regulates key inflammatory cytokines. Altogether, this part of my project not only deepen our knowledge regarding the impact of inflammation on mouse Müller cell behavior, it also highlights YAP as a key player in the crosstalk between inflammation and retinal regeneration

2013/2014
The Hippo signalling pathway is tumour suppressor cascade with a central role in the regulation of fundamental cellular biological processes, such as cell proliferation, apoptosis, organ size control and stem cell functions. The Hippo pathway transduces external signals that come to the cell into the nucleus, where it can control the expression of specific target genes, mainly involved in cell proliferation and differentiation. The Hippo pathway is an inhibitory pathway that control by phosphorylation and inhibition Yes-associated protein (YAP) coactivator, one of the two nuclear effectors of this signalling, involved in the regulation of proliferation and organ size. As consequence, deregulation of Hippo tumor suppressor pathway or hyperactivation of its downstream effectors is often associated with formation, development and tumour dissemination. Consistently, YAP is often over-expressed in a broad range of different tumours and it has aberrant activity in breast cancer as well as in several other human carcinomas. Up-regulation of YAP activity increases stem cell self-renewal in normal and cancer stem cells. In this work we describe the identification of a new hormonal-dependent layer for YAP regulation in breast cancer by the glucocorticoids and we analyze the mechanisms through which this regulation occurs. We found that Glucocorticoid Receptor (GR) binds directly the YAP promoter and induces the transcription of YAP mRNA after GC stimulation in cancer cells. Moreover, GC lead to efficient YAP de-phosphorylation and transcriptional activation, in a transcription-independent manner, by inducing actin cytoskeleton reorganization. Importantly, inhibition of the GR by means of RU486 (GR competitive antagonist) strongly blunted the expansion of the cancer stem cell pool in breast cancer cells by blunting the GR/YAP axis.
XXVII Ciclo
1987

Les maladies dégénératives de la rétine sont une des causes principales de cécité. Parmi différentes stratégies thérapeutiques actuellement étudiées, notre équipe s’intéresse au potentiel régénératif de la rétine. Une source cellulaire d'intérêt sont les cellules de Müller, principal type de cellules gliales de la rétine, capables de se réactiver en cas de dégénérescence et d’adopter certaines caractéristiques de cellules souches. Elles entrent alors dans un état appelé gliose réactive. Tandis que chez certaines espèces comme le poisson, elles permettent la régénération de la rétine, elles ont des capacités régénératives très limitées et inefficaces chez les mammifères. Une meilleure connaissance des mécanismes moléculaires régissant la gliose réactive des cellules de Müller est donc essentielle si l’on veut identifier des cibles thérapeutiques capables de stimuler le potentiel de régénération de ces cellules. Dans ce contexte, le but de mon projet de thèse a été d’étudier le rôle du co-facteur de transcription YAP dans la réactivation des cellules de Müller. Cette protéine est l’effecteur de la voie de signalisation Hippo, connue pour son implication dans la régulation des cellules souches et la régénération de certains organes.Dans un premier temps, nous avons réalisé une analyse transcriptomique qui a montré que la voie Hippo/YAP est une des principales voies dérégulées dans un modèle de dégénérescence rétinienne chez la souris. Nous avons ensuite montré que la protéine YAP est spécifiquement exprimée dans les cellules de Müller et que son expression et son activité transcriptionnelle sont augmentées au cours de la dégénérescence lorsque les cellules de Müller deviennent réactives. Ces données suggèrent pour la première fois un lien entre YAP et la gliose réactive dans la rétine. Par conséquent, dans un second temps, mon projet de thèse a consisté en l’étude fonctionnelle de YAP dans les cellules de Müller. Dans ce but, nous avons généré par croisements chez la souris un modèle inductible de délétion du gène Yap spécifiquement dans ces cellules. Ce modèle a permis de montrer qu’en absence de Yap en conditions physiologiques, plusieurs gènes spécifiques des cellules de Müller sont dérégulés, suggérant un dysfonctionnement de ces cellules. L’étude phénotypique a permis de révéler que ces dérégulations moléculaires conduisent à un vieillissement prématuré des cellules de Müller et à une baisse de la vision chez les souris âgées. Ces données suggèrent que YAP est requis pour le fonctionnement normal des cellules gliales de Müller. Nous avons ensuite examiné l’impact de la perte de Yap dans les cellules de Müller en conditions de dégénérescence des photorécepteurs. Une analyse transcriptomique a permis de montrer que différents aspects de la réponse moléculaire des cellules de Müller réactives sont affectés. Parmi les processus biologiques dérégulés, nous nous sommes intéressés à la régulation de la prolifération cellulaire. Nous avons montré que YAP est nécessaire à l’augmentation de l’expression de gènes associés à la réentrée dans le cycle cellulaire de la glie de Müller. Par ailleurs, nos résultats suggèrent que des composants de la voie de signalisation EGFR, connue pour son rôle central dans la réactivation des cellules de Müller, sont régulés par YAP.Dans l’ensemble, ces résultats révèlent l’importance de YAP (i) dans le fonctionnement des cellules de Müller en conditions physiologiques pour maintenir l’homéostasie rétinienne, et (ii) dans la régulation des processus de réactivation de ces cellules en conditions dégénératives. De plus, ces données permettent de proposer un modèle selon lequel YAP serait impliqué dans le contrôle de la réentrée des cellules de Müller dans le cycle cellulaire via une interaction avec la voie de signalisation EGFR. Ce travail a donc contribué à approfondir nos connaissances du réseau de signalisation impliqué dans la réactivation des cellules de Müller de la rétine des mammifères
Retinal dystrophies are one of the main causes of blindness. Among the different therapeutic strategies currently studied, our team is interested in the regenerative potential of endogenous retinal cells. A cellular source of interest are Müller cells, which are the main type of glial cells in the retina. These cells are able to reactivate in case of retinal degeneration and adopt various characteristics of stem cells. They enter a state called reactive gliosis. While in some species such as the fish, they allow the complete regeneration of the retina, they have very limited and ineffective regenerative capacities in mammals. Increasing our knowledge of the complex molecular response of Müller cells to retinal degeneration is thus essential for the development of promising new therapeutic strategies. In this context, the aim of my thesis project was to study the role of the co-transcription factor YAP in Müller cells reactivation. This protein is the main effector of the Hippo signaling pathway which is a crucial player in the field of stem cell biology and regeneration.As a first step, we performed a transcriptomic analysis, which revealed that the Hippo/YAP pathway is one of the main signaling deregulated in a mouse model of photoreceptor degeneration. In particular, we found that YAP is specifically expressed in Müller cells and strongly upregulated upon retinal degeneration, when these cells are reactivated. We thus uncovered for the first time a link between the Hippo/YAP pathway and reactive gliosis in the retina. Consequently, the second part of my thesis project was to undertake a functional study of YAP in Müller cells. For this purpose, we generated, by crossing, a mouse model allowing for Yap conditional knockout specifically in these cells. This model allowed us to show that Yap deletion leads to deregulation of several Müller cell specific genes. A phenotypic analysis revealed that these molecular deregulations lead to premature aging of Müller cells and visual defects in old mice. These results suggest that YAP is required for normal function of Müller glial cells. We then studied the impact of Yap deletion in Müller cells under degenerative conditions. A transcriptomic analysis revealed that various aspects of the molecular response of reactive Müller cells are affected in the absence of Yap. Among the deregulated biological processes, we focussed in particular in the regulation of cell proliferation. We found that YAP is required to trigger cell cycle gene upregulation that occurs in Müller glial cells following photoreceptor cell death. Furthermore, our results suggest that some components of the EGFR signaling pathway, which is known for its central role in the reactivation of Müller cells in pathological conditions, are regulated by YAP in Müller cells.Taken together, these results highlight the importance of YAP (i) in Müller cell function under physiological conditions to maintain retinal homeostasis, and (ii) in the regulation of Müller cell reactivation process under degenerative conditions. Moreover, these data allow us to propose a model in which YAP would be involved in the control of Müller glia cell cycle re-entry through its interaction with the EGFR signaling pathway. Therefore, this work has contributed to increase our knowledge of the signaling network involved in the reactivation of Müller cells in the mammalian retina

La polarité cellulaire est un déterminant essentiel dans le maintien de l’architecture tissulaire et la fonction de l’organe. Ainsi, la division cellulaire, la ciliogenèse, la prolifération, et la migration sont des évènements étroitement associés au processus de la polarisation cellulaire. L’altération de la polarité cellulaire contribue à la perte de l’intégrité des épithéliums et favorise le développement des cancers. La signalisation des lipides, telle que des phosphatidylinositols (PtdIns) joue un rôle vital dans la polarité apico-basale. Dans cette étude, nous avons développé des recherches pour mieux comprendre les mécanismes impliqués dans les effets de la phosphatase SHIP2 sur la polarité cellulaire. Nous avons pu démontrer que SHIP2 est impliquée dans la formation du site d’initiation de la formation de la lumière (AMIS) en régulant d’une part la contractilité acto-myosine induite par RhoA kinase et d’autre part YAP, un composant de la voie de signalisation Hippo. De plus, nous avons montré que l'inhibition de SHIP2 contribue à un défaut dans la formation de fuseau mitotique et dans le clivage de ce fuseau mitotique. La surexpression de SHIP2 induit une lumière large et des cils allongés attribuables à la diminution de l’expression de YAP, Aurora A et HEF1. Par contre, la diminution de l’expression de SHIP2 inhibe la formation des cils en provoquant la surexpression de YAP, Aurora A et HEF1 et ainsi l’apparition d’un phénotype multilumens. L’ensemble de nos travaux définissent un nouveau rôle de SHIP2 dans le maintien de l’intégrité et de l’homéostasie des cellules épithéliales. Nous avons aussi pu démontrer que l’expression de SHIP2 peut discriminer les différents cancers du foie (HCC, ICC et mixte) et que SHIP2 et Merlin/NF2, une protéine de la voie de signalisation Hippo, ont une forte expression dans le cholangiocarcinome (ICC) qui s’oppose à celle de YAP et de RhoA kinase
Cell polarity is critical caracteristic for the maintenance of tissue architecture. Cell division, ciliogenesis, cell proliferation and migration are events tightly associated to cell polarization processes. Alteration in cell polarity contributes to loss of epithelium integrity and enhances cancer development. Lipids signaling, such as phosphatidylinositol (PtdIns), play a vital role in apico-basal polarity. In this study, we developed researches to better understand mechanisms implicated the role of the phosphatase SHIP2 in cell polarity. We demonstrated that SHIP2 is implicated in formation of the apical membrane initiation site (AMIS) by regulating YAP, a component of Hippo pathway, and RhoA-dependant acto-myosin contractility. Furthermore, we demonstrated that inhibition of SHIP2 contributes to defect in the formation and cleavage of the mitotic spindle. Overexpression of SHIP2 induced a large lumen with long cilia due to a decrease in YAP, Aurora A and HEF1 luminal localization. On the contrary, down regulation of SHIP2 impaired cilia outgrowth by increasing Aurora A, HEF1 and YAP luminal localization with appearance of a multilumens phenotype. Thus, our results reinforced the role of SHIP2 in maintain of integrity and homeostasis of epithelial cells. In this study, we also demonstrated that expression of SHIP2 distinguished the different types of liver cancer (HCC, ICC and mixte), and that SHIP2 and Merlin/NF2 are overexpressed in ICC which is the opposite of YAP and RhoA expression

Les papillomavirus humains (HPV) constituent l’archétype des virus à ADN oncogènes. Les HPV muqueux à haut risque (principalement HPV16) sont les principaux agents étiologiques du cancer du col utérin. Les protéines virales E6 et E7 sont des acteurs cruciaux de la cancérogenèse induite par HPV. Nous avons construit une ressource composée de 600 ADNc codant les effecteurs humains du système ubiquitine-protéasome (UPS) et identifié de nouvelles cibles potentielles des protéines E6 et E7 en utilisant une méthode basée sur la complémentation protéique de la Gaussia princeps luciférase (GPCA). HPV16 E6 lie les motifs LxxLL présents dans E6AP et IRF3. Nous avons résolu la structure cristallographique des complexes E6/LxxLL de E6AP/p53 et E6/LxxLL de IRF3. Par ailleurs, nous avons montré que les HPV ciblent une nouvelle voie suppresseur de tumeurs, la voie Hippo, avec ses deux médiateurs clef YAP et TAZ. Nous avons généré une banque d’ADNc codant 265 domaines PDZ humains et identifié de nouveaux partenaires potentiels des protéines YAP/TAZ en utilisant la méthode GPCA. Les résultats obtenus permettent de mieux comprendre la biologie des HPV et leur pouvoir oncogène
The human papillomavirus (HPVs) are the archetype of DNA oncogenic viruses. High-risk mucosal HPVs (mainly HPV16) are the main causative agents of cervical cancer and are also involved in other cancers. HPV oncogenic properties are mainly due to the expression of E6 and E7 proteins. We built a resource composed of 600 cDNA encoding the human ubiquitin-proteasome system (UPS) effectors and identified novel E6 and E7 potential targets by using a method based on the complementation of the Gaussia princeps luciferase (GPCA). HPV16 E6 binds to specific LxxLL motifs present in E6AP and IRF3. We have solved the crystallographic structure of the E6/E6AP LxxLL/p53 and E6/IRF3 LxxLL complexes. Furthermore, HPV may target a novel tumour suppressor pathway, the Hippo signalling pathway with its two main mediators YAP and TAZ. We have built a cDNA library dedicated to the 265 human PDZ domains and identified news potential partners of YAP and TAZ proteins by using the GPCA. The results provide novel insights on HPV biology and their oncogenic property

Mechanical tension is an important regulator of cell proliferation, differentiation, migration and cell death. It is involved in the control of tissue architecture and wound repair and its improper sensing can contribute to cancer. The Hippo tumor suppressor pathway was recently shown to be involved in regulating cell proliferation in response to mechanical tension. The core of the pathway consists of the kinases MST1/2 and LATS1/2, which regulate the target of the pathway, the transcription co-activator YAP/ TAZ (hereafter referred to as YAP). When the Hippo pathway is inactive, YAP remains in the nucleus and promotes cell proliferation and stem cell maintenance. When the Hippo signaling pathway is turned on, MST1/2 phosphorylate and activates LATS1/2. LATS1/2 phosphorylates and inactivates YAP in the cytoplasm which is sequestered and degraded, stopping cell proliferation and promoting differentiation of stem cells. Mechanical forces are transmitted across cells and tissues through the cell-cell junctions and the actin cytoskeleton. However, the factors that connect cell-cell junctions to the Hippo signaling pathway were not clearly known. We identified a LIM domain protein called TRIP6 that functions at the adherens junctions to regulate the Hippo signaling pathway in a tension-dependent manner. TRIP6 responds to mechanical tension at adherens junctions and regulates LATS1/2 activity. Under high mechanical tension, TRIP6 sequesters and inhibits LATS1/2 at adherens junctions to promote YAP activity. Conditions that reduce tension at adherens junctions by inhibition of actin stress fibers or disruption of cell-cell junctions reduce TRIP6-LATS1/2 binding, which activates LATS1/2 to inhibit YAP. Vinculin has been shown to act as part of a mechanosensory complex at adherens junctions. We show that vinculin promotes TRIP6 inhibition of LATS1/2 in response to mechanical tension. Furthermore, we show that TRIP6 competitively inhibits MOB1 (a known LATS1/2 activator) from binding and activating LATS1/2. Together these findings reveal TRIP6 responds to mechanical signals at adherens junctions to regulate the Hippo signaling pathway in mammalian cells.

This thesis is divided in three sections; the main project is described in the first part, while additional projects are developed in two appendixes. In the main project we studied YAP, the downstream effector of the Hippo pathway, a transcriptional co-factor that plays a fundamental role in de-differentiation, cell proliferation and transformation. While its upstream regulation has been extensively studied, its role as transcriptional co-factor is still poorly understood. We show that YAP co-adjuvates the transcriptional responses of Myc oncogene to promote cell proliferation and transformation; when both YAP and Myc are overexpressed, YAP is recruited on genomic sites pre-marked by Myc, TEAD and active chromatin and potentiate the expression of cell cycle genes regulated by Myc. In addition, we show that YAP promotes cell de-differentiation by antagonizing in cis the expression of liver-specific genes controlled by HNF4A master regulator, thus providing a mechanism on how YAP can revert the phenotype of a differentiated hepatocyte into a progenitor cell. In the first appendix we explain the mechanism of BRD4 inhibition, a promising strategy for the treatment of Myc-driven tumors. The efficacy of this strategy relies on the control of transcriptional elongation mediated by BRD4 on gene promoters, independently of the downregulation of Myc oncogene. Although the inhibition of BRD4 causes its genome-wide displacement on promoters, the effects on transcription are restricted to a subset of sensitive genes. This specificity relies on the fact that while most genes compensate the drop in elongation caused by BRD4 inhibition with further recruitment of RNA Pol2 on promoters and maintain a proficient mRNA transcription, vulnerable genes are not able to promote these compensatory effects, because RNA Pol2 recruitment on these promoters is already maximized. Our results show how the impairment of elongation genome-wide can affect specific transcriptional programs. In the second appendix we describe a new web application, Chrokit, aimed at analyzing genomic data in a fast and intuitive way. Chrokit handles a set of genomic regions of interest and performs several tasks on them, such as selecting particular subsets, computing overlaps and visualize reads enrichment of specific chromatin features interactively. The application is multiplatform and can be run on dedicated servers to maximize computational power and provide accessibility to multiple users simultaneously.

2012/2013
The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate–YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.
XXVI Ciclo
1983

Dysregulated Hippo pathway signaling promotes the onset of aggressive cancers through the induced nuclear activity of yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) (YAP/TAZ). Uncontrolled nuclear YAP/TAZ activity evokes tumor-initiating properties in a range of epithelial-derived cancers, including oral and breast cancers, but their downstream targets and mechanisms of action are unclear. Recent studies have suggested that the pro-tumorigenic roles for YAP/TAZ relate to their convergence with growth factor signaling pathways. Based on these previous studies, I hypothesized that YAP/TAZ driven transcription contributes to carcinoma progression, and that cooperation with transforming growth factor β (TGFβ)-induced signals promotes aggressive oncogenic traits. In this thesis I show that dysregulated YAP localization precedes oral squamous cell carcinoma (OSCC) development, and that nuclear YAP/TAZ activity drives cell proliferation, survival, and migration in vitro, and is required for tumor growth and metastasis in vivo. Global gene expression studies in OSCC cells revealed that YAP/TAZ-mediated gene expression correlates with expression changes that occur in human OSCCs identified by “The Cancer Genome Atlas” (TCGA), many of which encode cell cycle and survival regulators. By exploring the relationship with growth factor signaling, I found that YAP/TAZ induce pro-tumorigenic events by converging with TGFβ-induced signals, particularly in breast cancer cells where TGFβ is known to promote metastatic properties. My observations indicated that YAP/TAZ are necessary for maintaining and promoting TGFβ-induced tumorigenic phenotypes in breast cancer cells, and that these phenotypes result from the cooperative activity of YAP/TAZ, the TEA domain family of transcription factors (TEADs), and TGFβ-activated SMAD2/3 in the nucleus. Genome-wide expression analyses indicated that YAP/TAZ, TEADs, and TGFβ-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by these complexes, such as the novel targets neuronal growth regulator 1 (NEGR1) and urothelial cancer associated 1 (UCA1), are necessary to maintain tumorigenic activity in metastatic breast cancer cells. Nuclear YAP/TAZ also cooperate with TGFβ signaling to promote phenotypic and transcriptional changes in non-tumorigenic cells to overcome TGFβ-mediated growth inhibition. This work thus defines novel roles for YAP/TAZ in cancer, offering molecular mechanisms that may be useful for identifying and targeting YAP/TAZ-driven cancers.

According to the Centers for Disease Control, cancer is one of the leading causes of death in the United States. Characterized as a disease that develops as a result of an unstable genome, cancer is known to arise from numerous spontaneous mutations in the DNA of cells. Recent evidence shows that cancer cells within tumors are not self-reliant; rather, they progress along with other cells in their surrounding environment. Tumor cells recruit neighboring cells that, like the cancer cells, also become deregulated, forming the tumor stroma that aids in tumor progression. Within the stroma, cancer-associated fibroblasts (CAFs) play a vital role in the progression of cancer. Recent studies have found an important link between increased matrix stiffness surrounding the tumor and the invasion of the tumor. Thus, it is proposed that as the matrix stiffens, the tumor takes on more aggressive phenotypes. The transcriptional regulators YAP and TAZ (YAP/TAZ), key effectors of the Hippo pathway, are known to respond and influence matrix stiffening. In stiff matrix environments YAP/TAZ accumulate in the nucleus, and can drive transcriptional events. CAF's from late stage breast cancers have been found to exhibit increased YAP expression and increased ability to remodel and stiffen the extracellular matrix. Whether YAP or TAZ in these CAFs influences the metastatic properties of tumor cells is unclear. The present study aims to establish a link between YAP/TAZ activity in CAFs and cancer migration and invasion. We hypothesized that high nuclear activity of YAP/TAZ in fibroblasts would lead to non-autonomous signals that increase epithelial migration, and conversely that signals originating from epithelial cells affect YAP regulation in fibroblasts. We obtained CAFs from oral squamous cell carcinomas (OSCC) at various stages, and interestingly found that when CAFs obtained from stage III and stage IV tumors were co-cultured with OSCC cells they had the ability to cause OSCC cell migration. This CAF-induced migration was dependent on YAP/TAZ in the CAFs, as YAP/TAZ knockdown repressed this crosstalk. To gain insight into the mechanisms driving this process, transwell migration assays were conducted using NIH-3T3 fibroblasts engineered to overexpress YAP, or mutants of YAP, in doxycycline-inducible manner. We found that expression of YAP in NIH-3T3 cells, particularly a nuclear-localized YAP mutant, promoted the ability for OSCC cells to migrate in co-culture experiments. Media conditioned from these cells was sufficient to recapitulate this phenotype, suggesting that secreted factors from these fibroblasts may act as a signal that promotes migration. This activity of YAP was dependent on the ability for YAP to bind to the TEAD transcription factors, a major mediator of YAP transcriptional activity. Together these results indicate that nuclear YAP activity in fibroblasts can modulate the migration of neighboring cancer cells, suggesting that YAP plays a key role in stroma-cancer crosstalk during cancer progression.

Indiana University-Purdue University Indianapolis (IUPUI)
The Hippo signaling pathway controls organ size by regulating cell proliferation, apoptosis, and cell differentiation. The Hippo pathway ultimately regulates the concentration of the coactivator yes-associated protein 1 (YAP1) in the nucleus, which binds the transcription factor TEA domains (TEAD), activating genes related to cell proliferation. In several cancers, increased YAP1 activity is linked to increased cellular proliferation, de-differentiation and survival to drive tumor progression and spreading. The development of inhibitors against YAP-TEAD binding are consequently a research topic of much interest. While several inhibitors of the YAP-TEAD interaction are reported, none possess both high specificity and anti-tumor activity. The work described here is based upon a collaboration with AtomWise, Inc to find a best in class competitive inhibitor of YAP-TEAD binding. AtomWise completed a computational screen of ten million compounds for potential binding to a TEAD pocket that is essential for interaction with YAP1. This master’s thesis encompasses both intracellular and biochemical validation of these compounds. Initially, a luciferase YAP/TEAD dependent reporter assay was used to identify compounds with potential intracellular activity. This data was compared with the action of these compounds on the metabolic activity (MTT assay) across three cancer cell lines. Three sulfonamide-based compounds, 4, 22, and 59 were identified as top 10 YAP/TEAD inhibitors. Six additional compounds were synthesized that combined specific moieties in these three compounds. Dose response curves for inhibition of TEAD reporter activity showed that compounds 22 and 59 exhibited both high inhibition and low IC50 values relative to the derivatives. Further, compound 22 also significantly reduced the levels of CTGF transcript (measured by RT-qPCR), a surrogate measure for endogenous YAP1: TEAD activity. Preliminary fluorescent polarization data also suggests that compound 22 inhibits the binding of a 5(6)-FAM labeled YAP1 peptide (residues 58-74) to a purified TEAD fragment. In this work, compounds 22 and 59 are found to inhibit TEAD dependent transcription and the growth of multiple cancer cell lines. In addition, promising preliminary biochemical data indicates that compound 22 may inhibit the interaction of YAP1 with TEAD.

Τα μηνιγγιώματα αποτελούν ιδιαίτερα συχνούς όγκους του Kεντρικού Νευρικού Συστήματος, όχι όμως επαρκώς μελετημένοι. Στην πλειοψηφία τους είναι καλοήθεις όγκοι (WHO grade I), όμως το 10% όλων των μηνιγγιωμάτων εμφανίζουν κακοήθη χαρακτηριστικά, όπως διήθηση των παρακείμενων ιστών, αυξημένα ποσοστά υποτροπής μετά από εξαίρεση, κ.α. Το σηματοδοτικό μονοπάτι Hippo είναι ένα διατηρημένο εξελικτικά μονοπάτι που εμπλέκεται σε διαδικασίες ρύθμισης του μεγέθους των οργάνων, κυτταρικού πολλαπλασιασμού, διαφοροποίησης και ανάπτυξης καρκίνου. Λίγα είναι γνωστά όσον αφορά το ρόλο του Hippo μονοπατιού στα μηνιγγιώματα. Σκοπός της παρούσας εργασίας είναι η μελέτη του Hippo μονοπατιού στα χαμηλής και υψηλής κακοήθειας μηνιγγιώματα. Χρησιμοποιήθηκαν 53 δείγματα από εξαιρεθέντα μηνιγγιώματα, 34 χαμηλής κακοήθειας και 19 υψηλής κακοήθειας, τα οποία μελετήθηκαν ανοσοϊστοχημικά ως προς την έκφραση των παραγόντων του Hippo CD44, YAP και TAZ. Παρατηρήσαμε στατιστικά σημαντική συσχέτιση μεταξύ της έκφρασης των CD44, ΥΑΡ και ΤΑΖ. Υπήρχε στατιστικά σημαντική συσχέτιση μεταξύ του βαθμού κακοήθειας και της έκφρασης των παραπάνω μορίων. Παρατηρήθηκε στατιστικά σημαντική διαφορά στην πυρηνική εντόπιση των ΥΑΡ και ΤΑΖ μεταξύ των υψηλής και χαμηλής κακοήθειας μηνιγγιωμάτων. Βάσει των παραπάνω αποτελεσμάτων, συμπεραίνουμε ότι το σηματοδοτικό μονοπάτι Hippo είναι απενεργοποιημένο στα υψηλής κακοήθειας μηνιγγιώματα με αποτέλεσμα οι μεταγραφικοί συνενεργοποιητές ΥΑΡ και ΤΑΖ να δρουν στον πυρήνα προάγοντας τον κυτταρικό πολλαπλασιασμό και διαδικασίες αντιαπόπτωσης. Αυτό, είναι ίσως εφικτό μέσω της δράσης του CD44 που αλληλεπιδρά με την μερλίνη, upstream ρυθμιστής του μονοπατιού. Απαιτούνται περαιτέρω μελέτες για τη διευκρίνηση του ακριβούς μηχανισμού.
Meningiomas are among the most common primary tumors of the Central Nervous System, but relatively understudied. The majority of meningiomas are benign tumors (WHO grade I), but almost 10% of all diagnosed meningiomas exhibit malignant features, such as invasiveness to the surrounding brain tissue, high recurrence rate, etc. Hippo signaling pathway is an evolutionary highly conserved protein kinase cascade involved in organ size control, cell proliferation and apoptosis, differentiation and cancer development. Very few are known about the role of Hippo pathway and meningiomas. In this study we attempt to find out whether Hippo path functions differently between high and low grade meningiomas. For this purpose paraffin embedded tissues obtained from 53 patients who underwent surgical removal of meningiomas were examined histoimmunologicaly. 34 out of 53 cases were low grade meningiomas and 19 out of 53 were high grade meningiomas. The paraffin sections were immunostained with CD44, YAP and TAZ antibodies, components of the Hippo signaling pathway. We observed statistically significant association between the expression of CD44, YAP and TAZ. There was a significant correlation between high grade meningiomas and expression of the examined factors. Moreover there was a significant difference in nuclear accumulation of YAP and TAZ between high and low grade meningiomas. Our findings suggest that in high grade meningiomas, Hippo pathway is inactivated and YAP and TAZ transcriptional co activators are able to insert nucleus and promote proliferation and antiapoptosis. This may be due to CD44 function, which interacts with merlin, an upstream regulator of the Hippo path. More experiments are required to verify the exact mechanism.

Mitosis is a critically important and time sensitive cellular process that proceeds rapidly, typically completing in 15-45 minutes. Mechanisms have evolved to measure the duration of mitosis, resulting in the identification of aberrant cells that spend too long in mitosis. If non-transformed cells undergo a mitosis that exceeds 90 minutes, then the resulting daughter cells activate a durable G1 arrest and cease proliferating. The underlying mechanism acting to time the duration of mitosis is unknown. Here, we demonstrate that cells activate the Hippo pathway upon entry into mitosis, which initiates degradation of the pro-growth transcriptional co-activators YAP and TAZ. Consequently, prolonged mitosis leads to decreased YAP/TAZ levels in the following G1, thus enforcing cell cycle arrest. We reveal that inactivation of the Hippo pathway, which is common in solid tumors, is sufficient to restore YAP/TAZ levels following a prolonged mitosis, and cells born from this prolonged mitosis can progress through the cell cycle. We also demonstrate that Hippo pathway inactivation alters cell fate decisions in response to mitotic arrest. Antimitotics (e.g. Taxol) have long been used to permanently arrest cells in mitosis, which frequently results in mitotic cell death. It has long been recognized that some cancer cells are resistant to antimitotics; this resistance can arise from cells escaping mitosis into the G1 phase in a process termed mitotic slippage. The mechanisms underlying these cell fate decisions are poorly understood. Here, we demonstrate that inactivation of the Hippo pathway promotes mitotic slippage and overall survival in cells treated with antimitotics by increasing antiapoptotic protein expression. Our data suggest that inactivation of the Hippo pathway may promote resistance to antimitotic therapies by favoring the survival and proliferation of cells that have experienced a prolonged mitosis. Interestingly, we find that restoring Hippo signaling to cancer cells that are resistant to antimitotic therapies sensitizes them to antimitotics and promotes mitotic cell death. Overall, we illuminate a broad role for Hippo signaling in determining cell fate during mitosis and identify a novel mechanism by which resistance to antimitotic therapies can arise.
2020-10-24T00:00:00Z

Le développement du tractus reproducteur femelle est issu de la coordination minutieuse de nombreuses voies de signalisation régulant les processus de prolifération, différenciation et d’apoptose cellulaire durant l’embryogenèse. Les voies Wnt et Hippo se démarquent à cet égard. L’activation de la voie Wnt, via des ligands spécifiques, participe à la stabilisation et l’augmentation de l’activité transcriptionnelle du coactivateur de transcription β-catenin. La voie Hippo, pour sa part, ne possède aucun de ligand spécifique. L’inactivation de la voie Hippo (via les kinases Lats1 et Lats2) entraine la stabilisation des coactivateurs de la transcription YAP/TAZ et l’augmentation de leur activité transcriptionnelle. Plusieurs évidences suggèrent notamment la possibilité de redondance fonctionnelle entre certains ligands de la voie Wnt, dont Wnt4 et Wnt5a, dans le développement du tractus reproducteur femelle. Cette avenue demeure toutefois peu étudiée. L’implication de la voie Hippo n’a pas été rapportée dans le développement du tractus reproducteur femelle. Toutefois, les nombreuses interactions rapportées dans la littérature entre les deux voies suggèrent un rôle méconnu de la voie Hippo. L’objectif de ce projet était donc d’élucider les rôles de Wnt4, Wnt5a, Lats1 et Lats2 dans le mésenchyme de Müller et le développement de l’utérus. Les résultats de notre première étude ont confirmé la fonction partiellement redondante de Wnt4 et Wnt5a dans le développement de l’utérus. Notre modèle est notamment caractérisé par des anomalies développementales ainsi qu’une perte de fonction utérine associée à des anomalies de décidualisation in vivo et une diminution de la viabilité des concepti. Les résultats de notre seconde étude ont confirmé les rôles redondants de Lats1 et Lats2 dans le maintien de la multipotentialité des cellules mésenchymateuses müllériennes. Une différenciation hâtive des cellules mésenchymateuses müllériennes en myofibroblastes via, entre autres, l’expression du gène cible Ctgf, a été observée. Nos résultats additionnels n’ont pu mettre en évidence une interaction potentielle entre les voies Wnt et Hippo pouvant expliquer l’apparition des phénotypes. Ces deux études permettent de confirmer certains rôles connus et d’établir de nouveaux rôles de ces voies dans le développement des canaux de Müller. Ils pourront aussi établir les fondements de modèles permettant l’étude de différentes pathologies utérines et l’identification de cibles thérapeutiques.
The development of the female reproductive tract arises from the coordination of numerous signaling pathways regulating processes such as proliferation, differentiation and apoptosis during embryogenesis. The Wnt and Hippo pathways are known to be involved in these processes. Wnt pathway activation, via its specific ligands, results in the stabilisation and increased transcriptional activity of β-catenin. The Hippo pathway does not possess any specific ligands. In contrast to Wnt, inactivation of the Hippo pathway (via Lats1 and Lats2 kinases) is required for the stabilization and increased activity of the transcriptional coactivators YAP and TAZ. The Wnt pathway is known to be involved in the development of the female reproductive tract. Further evidence also suggests the possibility of functional redundancy amongst certain WNT ligands such as Wnt4 and Wnt5a. The Hippo pathway is not known to be implicated in the development of the female reproductive tract. However, numerous interactions have been reported between both pathways, suggesting a possible unknown role of Hippo in that context. The objective of this project was to elucidate the roles of Wnt4, Wnt5a, Lats1 and Lats2 in the Müllerian mesenchyme and the development of the uterus. Results from our first study confirmed the partially redundant roles of Wnt4 and Wnt5a in the development of the uterus. Our model was notably characterized by developmental abnormalities and loss of uterine functions resulting in in vivo decidualization defects and loss of conceptus viability. Results from our second study confirmed the redundant roles of Lats1 and Lats2 in the maintenance of Müllerian mesenchymal cell multipotency. We observed premature differentiation of Müllerian mesenchymal cells into myofibroblasts in absence of both Lats1 and Lats2. These changes were in part due to the increased expression of the target gene Ctgf. Our additional results could not demonstrate any potential interactions between the Wnt and Hippo pathways that could explain the phenotypic changes. In conclusion, our studies confirmed and further discovered novel roles of these pathways in the development of the Müllerian ducts. These models could also lead to better understanding of the pathophysiology of certain uterine diseases and the discovery of potential therapeutic approaches.

The Hippo signaling pathway has been implicated in both mammalian organ size regulation, as well as tumor suppression. Specifically, the Hippo pathway plays a critical role regulating the activity of transcriptional co-activator, and downstream effector, Yes-associated protein (YAP), which modulates pro-proliferative transcriptional elements. Recent investigations have demonstrated that this pathway is activated in non-regenerating livers and its inhibition leads to liver overgrowth and tumorigenesis. The majority of the existing evidence regarding the role of the Hippo pathway in hepatocyte proliferation is based on in vitro studies and knock-out animal models. However, the role of the Hippo pathway during the natural process of liver regeneration, remains unknown. Here alterations in the Hippo signaling pathway were investigated, namely its interaction with angiomotin-like 2 (AmotL2) and Set7, during liver regeneration using a 70% rat partial hepatectomy (PH) model. Overall, results indicated no significant difference between AmotL2 levels in control and regenerating tissue at various time points during liver regeneration. No significant alterations in YAP methylation during liver regeneration were found compared to control tissue. In the end, results regarding the role of both AmotL2 and Set7 provided inconclusive evidence about their roles during the regenerative process. Given the role of the Hippo pathway in hepatocyte proliferation, a hypothesis was made that this pathway may play a role in pediatric liver tumors. YAP localization was evaluated using immunohistochemical analysis in tumor sections from patients with hepatoblastoma or hepatocellular carcinoma. Once again, the results were inconclusive at the time of the preparation of this manuscript due to technical difficulties in achieving satisfactory staining of the specimens. Further studies will be directed at elucidating the role of the Hippo pathway during liver regeneration as well as developing better conditions for the immunohistochemical staining of human liver specimens.