Le mésothéliome pleural malin (MPM) est une tumeur primitive de la plèvre, rare, très agressive, avec un pronostic sombre. Nous avons souhaité au cours de ce travail de thèse, identifier de nouveaux biomarqueurs du MPM en testant l’influence de l’inactivation des membres de la famille RASSF/Hippo sur la survie des 448 patients inclus dans l’essai clinique MAPS (IFCT-GFPC-0701). Nous souhaitions également comprendre quelles fonctions et signalisations essentielles à l’homéostasie cellulaire, auxquelles participe la voie de signalisation RASSF/Hippo, sont perturbées lors du processus de transformation des cellules mésothéliales. L’inactivation des membres de la voie a été étudiée par PCR spécifique de méthylation (MS-PCR) et leur influence sur la survie des 448 patients inclus dans l’essai clinique MAPS testée en analyse uni- et multivariée avant d’être validée par boostrap. D’autre part, nous avons mimé in cell, l’inactivation par ARN interférence de plusieurs membres de la voie Hippo dans des cellules de lignées mésothéliales humaines (MSTO-211H, H2452, H28 et H2052). Nous avons pu identifier plusieurs biomarqueurs du MPM : i) la kinase MST1 dont l’inactivation est un facteur de mauvais pronostic, ii) l’amphiréguline dont l’expression cytoplasmique est au contraire un facteur de bon pronostic et enfin iii) le CD44 dont l’expression élevée constitue un outil diagnostique du MPM. Les approches in cell, nous ont permis de démontrer que les altérations de la voie RASSF/Hippo induisent une activité inappropriée de l’effecteur terminal YAP : le moins bon pronostic des patients présentant une inactivation de MST1 s’explique ainsi par le fait qu’en régulant l’activité de YAP, MST1 contrôle la balance apoptose/prolifération et prévient l’invasion et la croissance sans adhésion. En son absence, ces processus cellulaires sont dérégulés. Ce travail démontre l’importance de l’axe CD44/RASSF1A/MST1 dans le contrôle d’une activité appropriée de YAP et de l’homéostasie des cellules mésothéliales. La compréhension des désordres cellulaires induits par la dérégulation de le voie RASSF/Hippo, désigne YAP comme cible thérapeutique potentielle chez les patients atteints de MPM et présentant des altérations de cette voie de signalisation. / Malignant pleural mesothelioma (MPM) is a rare, very aggressive, primary tumor with a poor prognosis. During this thesis, we wanted to identify new biomarkers of MPM by testing the influence of the RASSF/Hippo pathway inactivation on the survival of the 448 patients included in the clinical trial MAPS (IFCT- GFPC-0701). We also wanted to understand which functions and signals essential to cellular homeostasis, linked to RASSF/Hippo signaling pathway, are disturbed during the mesothelial cell transformation process. Inactivation of RASSF/Hippo members was studied by methylation-specific PCR (MS-PCR) and their influence on the survival of the 448 patients included in the MAPS clinical trial tested in uni- and multivariate analysis before being validated by bootstrap. We also mimed in cell, by RNA interference, several members of the Hippo pathway inactivation in human mesothelial cells lines (MSTO-211H, H2452, H28 and H2052). We have identified several biomarkers of MPM: i) MST1 kinase whose inactivation is a factor of poor prognosis, ii) amphiregulin whose cytoplasmic expression is on the contrary a factor of good prognosis and finally iii) CD44 whose high expression is a diagnostic tool for MPM. In cell we demonstrate that RASSF/Hippo pathway alterations induce an inappropriate activity of YAP, one Hippo end effector: the poorer prognosis of patients with inactivation of MST1 is thus explained by the fact that, by regulating YAP activity, MST1 controls the apoptosis/proliferation balance and prevents invasion and growth without adhesion from mesothelial cells. In its absence, these cellular processes are deregulated. This work finally demonstrates the importance of the CD44/RASSF1A/MST1 axis in controlling appropriate YAP activity and mesothelial cell homeostasis. The understanding of the cellular disorders induced by the of the RASSF/Hippo pathway deregulation designates YAP as a potential therapeutic target in patients with MPM and presenting alterations of this signaling pathway.

Recent evidence from cancer research indicates that lactate exerts a suppressive effect on innate immune responses in cancer. This study investigated the mechanisms by which lactate suppresses macrophage pro-inflammatory responses. Macrophages [Raw 264.7 and bone marrow derived macrophages (BMDMs)] were treated with LPS in the presence or absence of lactate. Pro-inflammatory cytokines, NF-κB and YAP activation and nuclear translocation were examined. Our results show that lactate significantly attenuates LPS stimulated macrophage TNF-α and IL-6 production. Lactate also suppresses LPS stimulated macrophage NF-κB and YAP activation and nuclear translocation in macrophages. Interestingly, YAP activation and nuclear translocation are required for LPS stimulated macrophage NF-κB activation and TNFα production. Importantly, lactate suppressed YAP activation and nuclear translocation is mediated by GPR81 dependent AMKP and LATS activation which phosphorylates YAP, resulting in YAP inactivation. Finally, we demonstrated that LPS stimulation induces an interaction between YAP and NF-κB subunit p65, while lactate decreases the interaction of YAP and NF-κB, thus suppressing LPS induced pro-inflammatory cytokine production. Our study demonstrates that lactate exerts a previously unknown role in the suppression of macrophage pro-inflammatory cytokine production via GPR81 mediated YAP inactivation, resulting in disruption of YAP and NF-κB interaction and nuclear translocation in macrophages.

No description available.

Melanoma, a malignant neoplasm of melanocytes, is the most lethal form of skin cancer. A majority of melanomas are driven by activating mutations in the kinase BRAF, which drives cellular proliferation through constitutive stimulation of the mitogen-activated protein kinase (MAPK) signaling pathway. Intriguingly, expression of oncogenic BRAF alone in vivo is insufficient to promote melanoma; rather, its expression leads to the development of benign nevi (moles) comprised of growth-arrested melanocytes. The acquisition of additional genetic or epigenetic changes is therefore critical for melanocytes to evade arrest and drive melanomagenesis, however the identity of these changes remains incompletely understood. Here we demonstrate that expression of oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway in vitro, which acts to limit melanocyte proliferation through the inhibition of the pro-growth transcriptional co-activators YAP and TAZ. Melanocyte-specific inactivation of Hippo signaling in vivo, via deletion of the Hippo kinases Lats1/2 alone, or in conjunction with oncogenic Braf expression, potently induces melanoma development in mice. Collectively, our data reveal that the Hippo tumor suppressor pathway represents an important barrier to melanoma development, and implicates YAP and TAZ as new therapeutic targets for the treatment of human melanoma.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21159号 / 農博第2285号 / 新制||農||1060(附属図書館) / 学位論文||H30||N5133(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 阪井 康能, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Through mutations in the genes TSC1 and TSC2, the genetic disorder Tuberous Sclerosis Complex (TSC) causes begin tumors to develop in different organs across the body. Of the many ways that this disorder can manifest, the brain is one of the most commonly affected organs in TSC. Mutations in TSC1 or TSC2 result in mTORC1 hyperactivation and can impact how the brain forms early in development. Most patients with TSC exhibit seizures and over half display some level of intellectual disability, highlighting the impact that mTORC1 hyperactivation can have on brain function and cognition. However, despite our understanding of the genetic cause of TSC, the mechanisms downstream of TSC1/TSC2 and mTORC1 that mediate TSC neuropathology are not well understood. Therefore, additional study of the cellular and molecular underlying the aberrant neurodevelopment found in TSC and other mTOR-overactivation disorders (collectively known as mTORopathies) is necessary for further understanding of these disorders. Of the pathways that have been identified to interact with mTORC1, there has been great interest in understanding the relationship between mTORC1 and Hippo-YAP/TAZ signaling. The Hippo pathway is an evolutionarily considered pathway that is crucial for regulating organ size through its control of the transcriptional co-activators YAP/TAZ. As exhibited through study of the murine brain, hyperactivation of YAP/TAZ causes changes in how the cortex develops, with several features overlapping with mTORC1 hyperactivation (including aberrant neuronal migration, changes in neuron structure, and increased progenitor proliferation). While the relationship between mTORC1 and YAP/TAZ has been explored in other systems, its connection in the brain has yet to be explored. In Chapter 1 of this dissertation, I first review how TSC affects cortical development as a whole by addressing what is known about the specific cell types and signaling pathways that are affected this disorder. Of the signaling pathways described, the Hippo- YAP/TAZ pathway is discussed in particular detail, addressing its role not only in the context of TSC and in terms of its interaction with mTORC1 signaling, but also in terms of its general role in cortical development. In discussing these studies, I describe the phenotypes seen in different mouse models and in the human brain, allowing for the identification of pathological features that are common between species and between different Cre lines. Following this initial review, I present our experimental aims, hypotheses, and experimental overview for this project in Chapter 2. In Chapter 3, I describe our investigation into the role of YAP/TAZ in the abnormal neurodevelopment that occurs in TSC. Through our analysis of human cortical tuber samples, I demonstrate that YAP/TAZ are elevated at the protein level and that two of their established target genes, CYR61 and CCN2, are elevated at the mRNA and protein levels. Having demonstrated that YAP/TAZ levels and activity are elevated in cortical tuber samples, I next went on to establish whether YAP/TAZ are similarly changed in our TSC animal model. Examination of Emx1-Cre driven Tsc2 cKO mice showed that the level of Yap/Taz were significantly elevated at E16.5. Having established that both YAP/TAZ levels are elevated in our animal model, I next sought to determine whether concurrent genetic manipulation of Yap/Taz in our Tsc2 cKO animals would reduce the severity of neuropathology seen in these mice. Triple conditional knockout (tcKO) of Yap/Taz/Tsc2 was sufficient to mitigate several features seen with mTORC1 hyperactivation in the brain, including the cortical thickness increases, abnormal neuronal migration in the cortex, hippocampal lamination defects, and hypomyelination found in their single Tsc2 cKO counterparts. Overall, these findings provide additional evidence that mTORC1 hyperactivation positively regulates YAP/TAZ. For the first time, this study describes elevation of YAP/TAZ in the brains of individuals with TSC and in the brains of a TSC mouse model. Furthermore, I provide evidence that reduction of Yap/Taz may have a beneficial effect on neuropathology in TSC, highlighting an area for future research in the development of novel therapeutics for this disorder. / Biomedical Sciences

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24791号 / 医博第4983号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 小林 恭, 教授 小濱 和貴 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

The Hippo signaling pathway was first discovered in Drosophila melanogaster and is involved in organ size control by regulating cell proliferation and apoptosis. This well conserved pathway is activated by various signal inputs, including cell-cell contact, mechanotransduction, and G-protein coupled receptors, with signals converging on the downstream effector protein Yap and its homologue Taz, which are transcriptional co-activators. When the Hippo pathway is activated, Yap/Taz are phosphorylated, leading to cytoplasmic retention and degradation, and diminishing their transcriptional activity. Yap has also been recently implicated as a potential oncogene, as it is upregulated and transcriptionally active in several tumor types. Furthermore, inhibiting Yap activity in various cancer models has been shown to revert cancer cells to a normal phenotype. Although the role of Yap has been described in several organ systems, there is a paucity of information about the function of Yap in the central nervous system. I investigated the function of Yap/Taz in the murine cerebellum to determine its significance during normal development and a potential role for Yap/Taz in medulloblastoma, a tumor that arises in the cerebellum. In Chapter 2, I describe the expression pattern of Yap from embryonic through adult stages in mice, and demonstrate the functional significance of Yap/Taz in different cell populations using conditional knockout mouse models. I show that Yap plays a significant role in cell fate determination as well as in cerebellar foliation: Yap is highly expressed in the ventricular zone and is required for the proper formation of ependymal cells, and is also strongly expressed in Bergmann glia (BG) during early developmental stages, where Yap, together with Taz, plays a significant role in cerebellar foliation. Furthermore, Yap/Taz-deficient BG exhibit migrational defects, as their cell bodies can be found mislocalized to the molecular layer (ML), rather than remaining tightly associated with Purkinje Cells (PCs) in the PC layer. BG support the health of PCs, and severely defective BG positioning eventually leads to a loss of PCs. However, although Yap is highly expressed in granule neuron progenitors (GNPs) during the rapid postnatal expansion stage, it does not appear to play a major role in proliferation of these cells as conditionally knocking-out Yap/Taz in GNPs does not alter their proliferative capacity. Our observations demonstrate that in the cerebellum, Yap has a novel function in glia that is required for the development of normal foliation and organization, but plays a minimal role in GNP proliferation. Importantly, I also show that the reduction of sphingosine-1-phosphate G-protein-coupled receptor (S1P1) signal transduction activates the upstream kinase Lats with concomitant increases of phosphorylated Yap as well as a reduction of the known Yap target connective tissue growth factor (CTGF). This study identifies a novel function of Yap/Taz in cerebellar glia that is required for the development of normal foliation and laminar organization with sphingosine-1-phosphate (S1P) signaling as a potential extracellular cue regulating Yap activity during cerebellar development. In Chapter 3, I present further support for the finding that Yap/Taz are not required for GNP proliferation in vivo by discussing the failure of Yap/Taz loss to rescue the Sonic-hedgehog (Shh) mediated medulloblastoma phenotype, in which GNPs are considered to be the tumor cell of origin. Furthermore, I provide evidence suggestive of a tumor suppressive function of Yap/Taz in the cerebellum. Together, previously unknown functions of Yap in the developing and malignant cerebellum are described, providing a foundation for future studies of Yap in the central nervous system (CNS). / Biomedical Sciences