Macroautophagy (hereafter referred to as autophagy) is a highly conserved cellular process that promotes cytoplasmic homeostasis via lysosomal degradation of proteins and organelles. Dysfunctional autophagy occurs in numerous human pathologies, including neurodegeneration and cancer. Vinexin (encoded by SORBS3) is a physiologically important adaptor protein for two main reasons: 1. SORSB3 mRNA expression increases in normal human brain ageing, 2. SORBS3 is a candidate tumour suppressor in hepatocellular carcinoma (HCC). This dissertation builds on published data from an siRNA screen for autophagy regulations under basal conditions, which indicates vinexin knockdown upregulates autophagy. I replicate this finding in multiple cell lines, before characterising the impact of siSORBS3 treatment on autophagy; autophagosome biogenesis is increased, while flux through the autophagy pathway remains intact. Having excluded several possible mechanisms suggested by the literature, I focus on the transcriptional coactivators YAP and TAZ. The rationale here is: 1. YAP/TAZ activity is implicated in autophagy, 2. YAP/TAZ and vinexin are both linked to HCC. My data show that YAP/TAZ transcriptional activity is upregulated upon vinexin depletion. Moreover, increased autophagy following siSORBS3 treatment requires YAP and TAZ. A key focus of this dissertation is the mechanism by which vinexin knockdown upregulates YAP/TAZ and hence, autophagy. This centres on altered actin cytoskeleton dynamics; an increase in F-actin structures appears to compete with YAP/TAZ for binding to angiomotins, established sequesterers of YAP/TAZ in the cytosol. In this way, siSORBS3 treatment facilitates YAP/TAZ nuclear localisation and consequent transcriptional activity. Angiomotin overexpression therefore ameliorates the increase in autophagy caused by vinexin depletion. Published RNA sequencing data is used to confirm that SORBS3 mRNA expression increases in normal brain ageing, not only in the frontal cortex (as previously published), but also in the hippocampus. This sits alongside decreased expression of core autophagy genes in both tissues. Accordingly, vinexin could contribute to the decline in autophagic potential thought to occur in normal brain ageing. With regards to SORBS3 as a candidate tumour suppressor in HCC, I show that stably re-expressing vinexin in a HCC cell line downregulates YAP/TAZ and hence, autophagy. These cells also show reduced clonogenicity. My data therefore support the hypothesis that SORBS3 is a tumour suppressor in HCC; YAP and TAZ are well-known to increase proliferation and resistance to apoptosis, while autophagy can enable tumour cells to survive stressors such as nutrient starvation. The conclusions of this dissertation are that vinexin depletion upregulates autophagy in a YAP/TAZ-dependent manner and that this has physiologically important implications, especially with regards to HCC.

La voie Hippo, découverte chez la Drosophile et conservée chez les mammifères, a été identifiée comme un élément essentiel dans le contrôle de la taille des organes. Cette cascade de kinases régule la phosphorylation de l’effecteur terminal YAP (ou de son paralogue TAZ), un coactivateur transcriptionnel reconnu comme oncogène. Sa fonction est médiée par sa translocation nucléaire et son interaction avec les facteurs de transcription TEAD, pour former le complexe YAP-TEAD qui active l’expression des gènes cibles responsable de la prolifération cellulaire et de la croissance des organes. La surexpression des protéines YAP/TAZ/TEAD dans de nombreux cancers perturbe l’équilibre de la voie Hippo et favorise la formation du complexe protéique causant une hyperprolifération et la propagation des cellules cancéreuses. Inhiber cette interaction protéine-protéine est une cible thérapeutique prometteuse pour concevoir de nouveaux anticancéreux. Dans cette optique, le laboratoire a considéré deux stratégies. La première consiste à cibler la protéine YAP en synthétisant des dipyrrines, représentant des fragments de la Vertéporfine dans le but de définir le motif minimal requis pour conserver l’activité biologique. Une seconde approche implique la synthèse de ligands de TEAD capable de se positionner au sein de l’interface 3. Basée sur des études de modélisation moléculaire, une famille avec un noyau central triazolique a été optimisée pour établir des relations structure-activité. Les molécules synthétisées sont actuellement en cours d’évaluation, grâce à la mise au point des tests biologiques et physicochimiques, et les premiers résultats ont permis d’identifier un composé prometteur. / The Hippo pathway, firstly described in Drosophila and highly conserved in mammals, has been demonstrated to play a crucial role in the organ size control. This kinase cascade regulates the phosphorylation of the downstream effector YAP (or its paralogue TAZ), a transcriptional coactivator with oncogenic activity. Its function is mediated by its nuclear translocation and interaction with the transcriptional factor TEAD, to form YAP-TEAD complex which activates the genes expression in charge of cell proliferation triggering organ growth. Overexpression of YAP/TAZ/TEAD protein in several cancers disrupts the Hippo pathway balance and urges on YAP-TEAD complex formation causing excessive proliferation and cancer development. Inhibiting this protein-protein interaction is thus a promising therapeutic target for the design of new anti-cancer drugs. In this goal, the laboratory has considered two strategies. The first one consists in targeting the YAP protein by synthesizing dipyrrins, representing Verteporfin fragments to define the minimal requirement yielding the expected biological activity. A second approach involves synthesizing TEAD ligands able to fit in specific interface 3. Based on molecular modeling, a triazole scaffold family was optimized to establish structure-activity relationship. Thanks to the biological and binding tests development, synthesized molecules evaluation is still in progress and the present first results have already allowed identifying a promising compound.

Indiana University-Purdue University Indianapolis (IUPUI) / The growth of mammary epithelial cells is regulated by interactions with neighboring cells and by exposure to soluble factors including hormones and growth factors. These cues are integrated within the cell, perpetuating changes onto the organization of the actin cytoskeleton, resulting in altered transcriptional programs. Rho family GTPases regulates actin dynamics that facilitate transcriptional reprogramming. In particular, RhoA induces the formation of actin stress fibers to promote the transcriptional co-activator YAP1 to translocate from the cytosol into the nucleus. There, it co-activates TEAD family transcription factors to drive the expression of pro-growth and survival genes. Rho family members are activated by guanine exchange factors (GEF) and inhibited by GTPase activating proteins (GAP). Here, we determined the relative effects of expression of 67 RhoGEFs and RhoGAPs on the activation of TEAD. This revealed that regulator of G-protein signaling (RGS) domain containing ArhGEF1, ArhGEF11 and ArhGEF12 all promoted YAP1 dependent activation of TEAD. These RhoGEFs mediate signaling from heptahelical receptors that are stimulated by lipid mitogens to activate the heterotrimeric G-proteins Gα12 and Gα13. Consistently, loss of expression of ArhGEF12 and to a lesser degree ArhGEF11 prevented actin stress fiber accumulation and activation of YAP1 mediated signaling by serum. Conversely, several complementary experiments revealed that ArhGEF1 dominantly limits Gα13 selective activation of YAP1 and the mitogen activated protein kinase (MAPK) cascades. Furthermore excessive Gα13 activity results in both high levels of filamentous actin and arrest cells in the G1/0 phase of the cell cycle. This is likely due to the systemic inhibition of cell cycle promoting signaling and a loss of protein translation. Further, YAP1 was found to be essential for the survival of ArhGEF1 silenced cells. Together, these studies define a circuit whereby the rgRhoGEFs regulate Gα 12/13-RhoA signaling flux to regulate cellular growth that is promoted by serum factors.

The Hippo pathway is frequently deregulated in human cancers, but mutations and deletions of core signalome members are rare, suggesting that our understanding of its upstream regulators remains incomplete. A focused RNAi-based kinome screen identified novel candidate regulators of Hippo signaling, including STK25 and MST4. Here, we characterize the kinase STK25 as a novel upstream activator of LATS signaling. Depletion of STK25 was found to significantly reduce YAP phosphorylation in response to Hippo-activating stimuli, with consequent increases in YAP/TAZ activity and increased proliferation and resistance to cell cycle arrest. Mechanistically, STK25 activates LATS independently of the canonical MST/MAP4K axis, wherein STK25 directly promotes the phosphorylation of the LATS kinase activation loop in the absence of a preceding hydrophobic motif phosphorylation event. This differentiates STK25 from all other identified Hippo kinases to date, which may explain why singular loss of this kinase cannot be compensated for by the presence of other Hippo kinases. We also find that loss of STK25 increases YAP/TAZ signaling in vivo and that this promotes organ overgrowth in murine models. Interestingly, STK25 is frequently focally deleted in a spectrum of human cancers, suggesting that its loss might represent a way by which cancer cells functionally inactivate Hippo signaling. We also report that STK25 may be a novel regulator of mTOR signaling, as loss of STK25 hyper-activates mTOR signaling in response to amino acids and growth factors, but not to energy stresses. Interestingly, we find that MST4, a kinase closely related to STK25, appears to have highly context-specific Hippo regulatory functions; loss of MST4 was found to modulate Hippo signaling only in non-polarized cell lines, suggesting that polarity-responsive subcellular localization of MST4 may dictate its ability to interact with Hippo signaling. Lastly, we describe a novel role for Hippo signaling as a surveillance system for abnormal prolongation of mitosis, in which the LATS kinases regulate cell fate following abnormal mitosis via its control over p53-p21 signaling and YAP/TAZ signaling. This work thus identifies new roles and mechanisms by which kinases interact in the context of this major tumor suppressor pathway to control cellular processes critical to physiologic homeostasis. / 2021-10-07T00:00:00Z

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23334号 / 生博第452号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 渡邊 直樹, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

A sinalização da matriz extracelular (MEC) é essencial para a determinação do destino e comportamento de células epiteliais da glândula mamária. Entretanto, pouco é conhecido sobre os mecanismos moleculares envolvidos nesse processo. A via Hippo, uma cascata de sinalização que participa da regulação de diversos comportamentos celulares, incluindo o tamanho de órgãos, parece ser uma importante candidata a mediadora sinalização da MEC. Resultados preliminares do laboratório indicam que a arquitetura tecidual e a membrana basal, componente da MEC de epitélios e outros tecidos, influenciam a localização, concentração e atividade de YAP, uma proteína efetora da via Hippo, em células epiteliais mamárias. Neste contexto, o objetivo deste trabalho foi identificar as proteínas que interagem com Yap (ortólogo de YAP em camundongo) nas células epiteliais da glândula mamária em resposta à membrana basal. Foram utilizadas células EpH4, uma linhagem mamária não-tumoral murina, como modelo de diferenciação funcional e formação de ácinos em um ensaio de cultura tridimensional (3D). O tratamento de estruturas multicelulares 3D pré-formadas em placas nãoadesiva com uma matriz rica em laminina (lrECM) alterou a localização e o padrão subcelular de Yap, assim como a expressão gênica de membros da via Hippo e dos alvos de Yap, mas não alterou a expressão das proteínas da via em nível de proteína. O ensaio de co-imunoprecipitação (CoIP) seguida de análise por espectrometria de massas identificou um conjunto diferencial de proteínas que interagem com Yap na fração citoplasmática de células EpH4 cultivadas na ausência ou na presença de lrECM em um modelo de ECM-overlay. Uma análise realizada junto à database KEGG Pathways revelou que os possíveis interagentes Yap nas células cultivadas não-tratadas com lrECM participam de processos relacionados à proteólise mediada por ubiquitina, enquanto nas células expostas à lrECM os possíveis interagentes estão associados a processos metabólicos e são especialmente proteínas-chave do metabolismo de lipídios. A busca na plataforma de redes de interação STRING não identificou trabalhos que destaquem a interação de Yap com estas proteínas. A plataforma Vizit indica a participação de Yap em processos relacionados à síntese e atividade de lipídios e hormônios, o que reforça as evidências de que está pode ser uma nova função de Yap ainda não explorada em detalhes. A fim de se obter resultados complementares à CoIP, padronizamos o ensaio de identificação por biotinilação dependente de proximidade (BioID) em células embrionárias de rim humano da linhagem 293FT. As proteínas isoladas por pulldown foram identificadas por espectrometria de massas e uma análise junto à database Gene Ontology indicou que os possíveis interagentes de Yap nestas células são em sua maioria proteínas relacionadas à via Hippo, o que reforça a robustez do ensaio. Nós pretendemos transpor este sistema para as células EpH4. A expectativa é que, em conjunto, estes resultados nos orientem em projetos futuros para compreender os mecanismos de sinalização da MEC na morfogênese e diferenciação da glândula mamária. / Extracellular matrix (ECM)-signaling is crucial for determination of epithelial cell fate and behavior in the mammary gland. However, little is known about the molecular mechanisms involved in these processes. The Hippo pathway, a signaling cascade involved in the regulation of several cellular processes, including organ size, seems to be an important candidate as a mediator of this signaling. Our preliminary results indicate that the tissue architecture and the basement membrane, an ECM component of epithelia and other tissues, influence the location, level and activity of YAP, an effector of the Hippo pathway. In this context, the goal of this work was to identify the proteins that interact with Yap (ortholog of YAP in mouse) in mammary epithelial cells in response to the basement membrane. We used EpH4 cells, a nontumoral murine mammary cell, in a functional differentiation and acini-forming in tridimensional (3D) culture assay. Treatment of 3D multicellular structures pre-formed on nonadhesive plates with a laminin-rich extracellular matrix (lrECM) altered the subcellular localization and pattern of Yap, as well as gene expression of Hippo pathway proteins and Yap targets, but did not altered the expression of the pathway members at the protein level. Coimmunoprecipitation (CoIP) followed by mass spectrometry analysis identified a differential set of proteins interacting with Yap in cytoplasmic fractions of EpH4 cells in the absence or presence of lrECM in an ECM-overlay culture model. An analysis performed with the KEGG Pathways database revealed that putative Yap interactors in non-treated cells participate in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to lrECM Yap interactors are associated to metabolic processes and are mainly key-proteins of metabolism of lipids and carbohydrates. A search in interaction networks platform STRING did not identify previous works that showing the interaction of Yap with these proteins. Vizit platform indicated the participation of Yap in processes related to the synthesis and activity of lipids and hormones, which reinforces the evidences that Yap can play a novel poorly explored role. To obtain complementary results to CoIP, we devised the proximity-dependent biotinylation identification (BioID) assay on embryonic renal cells of 293FT cell line. Pulldown-isolated proteins were identified by mass spectrometry and an analysis performed with Gene Ontology database revealed that putative Yap interactors are Hippo pathway-related proteins, which reinforces the robustness of the assay. We intend to transpose this system to the EpH4 cells. We expect that, together, these results will guide us in future projects to understand the signaling mechanisms of ECM in mammary gland morphogenesis and differentiation.

Contrairement aux mammifères adultes, la rétine des amphibiens possède la particularité de croître durant toute la vie de l'animal grâce à l'activité continue d'une population de cellules souches localisée au sein d'une niche bien délimitée, la zone marginale ciliaire (ZMC). Ce modèle offre ainsi la possibilité d'étudier in vivo les mécanismes moléculaires à l'origine du maintien et de la prolifération des cellules souches neurales à des stades post-embryonnaires. Dans ce but, l'identification et la caractérisation des différentes voies de signalisation présentes au sein de la niche biologique des cellules souches rétiniennes est une première étape indispensable. Mon projet de thèse a été divisé en deux objectifs principaux: l'étude des interactions entre les voies Wnt et Hedgehog au sein de la ZMC chez le xénope et la réalisation de l'étude fonctionnelle de Yap, l'effecteur principal de la voie de signalisation Hippo dans ce modèle. Par des approches génétiques et pharmacologiques, la première partie de ce projet a permis de mettre en évidence un antagonisme inattendu entre les signaux Wnt et Hedgehog au sein de la ZMC qui régule l'activité proliférative des cellules souches et des progéniteurs rétiniens. Ce travail nous a conduit à proposer un modèle dans lequel ces deux voies réguleraient la balance prolifération/différenciation dans la rétine post-embryonnaire. Dans un deuxième temps, les expériences de gain et de perte de fonction du gène Yap ont montré que ce dernier joue un rôle essentiel dans la régulation du programme temporel de la phase de réplication de l'ADN des cellules souches rétiniennes. En effet, l'inhibition de Yap entraîne une importante réduction de la durée de la phase S du cycle cellulaire associée à une instabilité génomique. Une surexpression de c-Myc et de la voie p53-p21 semble impliquée dans ce phénotype. Nos travaux nous ont également permis d'identifier un nouveau partenaire de YAP, le facteur de transcription PKNOX1. L'ensemble de ces données nous a ainsi conduit à proposer un modèle selon lequel le complexe YAP/PKNOX1 pourrait être nécessaire au bon déroulement de la phase de réplication des cellules souches, indispensable à la maintenance de l'intégrité du génome de ces cellules et de leur descendance. / In contrast to the adult mammals, the retina of amphibians shows continuous growth during adulthood through active neural stem cells localized in the defined niche called ciliary marginal zone (CMZ). This model offers an exceptional tool to study in vivo the molecular mechanisms involved in the maintenance and proliferation of neural stem cells during post-embryonic stages. In this order, the identification and the characterization of the signaling pathways acting in biological retinal stem cell niche is an essential step.My PhD research was divided in two main parts: the study of the interaction between the Wnt and Hedgehog pathways within the CMZ and the functional study of Yap, the downstream effector of the Hippo pathway in this model. By using genetic and pharmacological tools, the first part of this project demonstrated an unexpected antagonism between the Wnt and the Hedgehog signaling in the CMZ that regulates proliferative activity of retinal stem and progenitor cells. In this article, we propose a model in which an antagonistic interplay of Wnt and Hedgehog pathways may regulate the balance proliferation/differentiation in the post-embryonic retina. Second, gain and loss of function experiments of Yap have shown that this factor plays a key role in the regulation of temporal replication of DNA retinal stem cells. Indeed, inhibition of Yap leads to strong reduction of the S-phase length during the cell cycle associated with genomic instability. c-Myc and p53-p21 overactivation seems to be involved in this phenotype. This work also allowed us to identify a novel YAP partner, the transcriptional factor PKNOX1. We indeed propose a model in which the YAP/PKNOX1 complex may be required for the successful convening of the replication phase on stem cells, essential for the maintenance of genome integrity on the cells and their progeny.

Ph. D. Thesis--University of Oregon, 1998. / Bibliogr. p. 112-124.

La sclérose tubéreuse complexe (TSC) est une maladie génétique caractérisée par une croissance des hamartomes dans différents organes y compris le cerveau, les reins, les poumons, la peau et le cœur. Ces lésions sont des sources de morbidité et de mortalité chez les patients TSC, car ils peuvent provoquerl’ épilepsie, l’autisme, le retard de développement et l’insuffisance rénale et pulmonaire. Les causes connues de TSC sont la perte de la fonction et les mutations des gènes TSC1 et TSC2. La majorité des lésions TSC contient plusieurs types cellulaires de la lignée mésenchymateuse, comme dans le cas des angiomyolipomes, l’lymphangioleiomyomatose et les angiofibromes. Un type unique de cellules épithélioïdes périvasculaires nommé (PEC) est constamment présent dans les lésions de TSC mésenchymateuses, comme angiomyolipomes et lymphangioleiomyomatose, basant sur les caractérisations morphologiques et l'expression des marqueurs communs mélanocytaires et myogéniques. Par conséquent, ces lésions sont officiellement classées, ainsi que d'autres tumeurs, comme PEComes. Leur origine cellulaire et les mécanismes moléculaires impliqués dans la pathologie restent à élucider. Ici, nous avons généré un modèle souris mosaïque TSC1 knockout qui développe des lésions rénales mésenchymateuses récapitulant périvasculaire épithélioïde cellules tumorales humaines (Pecoma) observés chez les patients TSC. Nous avons identifié YAP, le co-activateur transcriptionnel de la voie Hippo, a été régulée d'une manière mTOR-dépendante dans les lésions rénales de notre TSC1 knockout souris et les échantillons de l’angiomyolipome humaines. L'inhibition de YAP avec des outils génétiques ou pharmacologiques atténue considérablement la prolifération et la survie des cellules nulles TSC1 in vivo et in vitro. En outre, l’accumulation de YAP dans les cellules déficientes TSC1 / TSC2 pourra être dû à la dégradation de la protéine altéré par le système de l’autophagosome / lysosome. Ainsi, la régulation de YAP par mTOR et l'autophagie est un nouveau mécanisme de contrôle de la croissance, l'activité de YAP correspondant à la disponibilité des éléments nutritifs dans les conditions de croissance permissives. Il pourra servir comme une cible thérapeutique potentielle pour TSC et d'autres maladies avec une activité de mTOR dérégulée. / The Tuberous Sclerosis Complex (TSC) is a genetic disease characterized by growth of hamartomas in different organs including brain, kidney, lung, skin, and heart. These lesions are sources of morbidity and mortality in patients with TSC, as they may cause intractable epilepsy, autism, developmental delay, renal and pulmonary failure. Known causes of TSC are loss of function mutations in TSC1 and TSC2 genes. The majority of TSC lesions contain multiple cell types of the mesenchymal lineage, as in the case of angiomyolipomas, lymphangioleiomyomatosis and angiofibromas. A unique cell type named perivascular epithelioid cell (PEC) is constantly present in mesenchymal TSC lesions, such as angiomyolipomas and lymphangioleiomyomatosis, basing on morphological features and the common expression of melanocytic and myogenic markers. Therefore, these lesions are officially classified, along with other tumors, as PEComas. Their cell of origin and the molecular mechanisms underlying their pathogenesis remain poorly defined. Here we generated a novel mosaic Tsc1 knockout mouse model which develop renal mesenchymal lesions recapitulating human Perivascular Epithelioid Cell tumor (PEComa) observed in TSC patients. We identified YAP, the transcriptional coactivator of Hippo pathway, was upregulated in both renal lesions of TSC mouse model and human angiomyolipoma samples in a mTOR-dependent manner. Inhibition of YAP with genetic or pharmacological tools greatly attenuates the proliferation and survival of Tsc1 null cells in vivo and in vitro. Futhermore, we found YAP accumulation in TSC1/TSC2 deficient cells is due to impaired degradation of the protein through the autophagosome/lysosome system. Thus the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth permissive conditions. It may serve as a potential therapeutical target for TSC and other diseases with dysregulated mTOR activity.

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.