Les tumeurs mammaires canines (CMT) sont les plus fréquents néoplasmes chez les chiennes intactes. Aucun consensus n’existe, actuellement, concernant le protocole thérapeutique des tumeurs invasives ou métastatiques. Des études récentes démontrent l’implication du dérèglement de la voie Hippo dans le développement et la progression des CMTs. Les statines, en inhibant la geranylgeranylation (GGylation) protéique, activent la voie Hippo et suspendent les effets transcriptionnels de YAP et TAZ. Cette étude évalue le ciblage thérapeutique de la voie Hippo avec deux statines (atorvastatine et fluvastatine) sur deux lignées cellulaires de CMT (CMT9 et CMT47). Les deux statines ont démontré une cytotoxicité pour CMT9 et CMT47 avec de IC50 de 0,95 µM et 23,5 µM respectivement. Les statines ont également diminué les niveaux des protéines effectrices de la voie, YAP et TAZ, ainsi que les niveaux d’ARNm de trois gènes cibles de YAP-TAZ/TEAD connus pour leur rôle dans la progression du cancer du sein et la chimiorésistance : CYR61, CTGF et RHAMM. De plus, la migration cellulaire de CMT47 et la croissance indépendante de l’ancrage des deux lignées ont été inhibées en présence de statines mais pas l’invasion de la matrice extracellulaire. Enfin, les analyses FACS ont montré une augmentation de l’apoptose et un arrêt du cycle cellulaire. Nos résultats suggèrent que les statines activent la voie Hippo et ciblent différents mécanismes moléculaires et cellulaires dans les CMT in vitro. Les statines agissent sur la voie Hippo et pourraient représenter une approche innovante pour le traitement des tumeurs mammaires canines invasives ou métastatiques. / Canine mammary tumours (CMTs) are the most common neoplasms in intact bitches, and no effective chemotherapeutic options are available for highly invasive and metastatic tumors. Recent studies show the potential involvement of dysregulated Hippo signaling in CMT development and progression. Protein geranylgeranylation (GGylation) is an important post-translational modification for many signaling molecules, and its blockade with statins has been shown to inhibit YAP/TAZ-mediated transcriptional activity via activation of the Hippo pathway. In this study, we sought to determine if protein GGylation represents a valid pharmacological target in CMTs. Two CMT cell lines (CMT9 and CMT47) were evaluated for their sensitivity to atorvastatin and fluvastatin. Results demonstrated statins to be cytotoxic to both cell lines, with IC50 values ranging from 0.95 µM to 23.5 µM. In addition, both statins lowered Hippo pathway effector proteins YAP and TAZ and reduced the mRNA levels of key transcriptional target genes known to be involved in breast cancer progression and chemoresistance (CYR61, CTGF and RHAMM). Moreover, both statins effectively inhibited cell migration in CMT47 and anchorage independent growth in CMT9 and CMT47, but did not modulate matrix invasion. Finally, our FACs results showed that both statins increase apoptosis and promote cell cycle arrests. Taken together, our results indicate that statins activate the Hippo pathway in CMTs and modulate several aspects of CMT’s molecular and cellular behaviours. These findings suggest that targeting the Hippo pathway with statins could be a novel approach for the treatment of invasive, metastatic or inoperable canine mammary gland cancers.

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.

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.

Trabalho de investigação desenvolvido na Universidade do Porto. Instituto de Ciências Biomédicas Abel Salazar, e no National Institute for Materials Science(Tsukuba, Japan) / Tese de mestrado integrado. Bioengenharia (Biotecnologia Molecular). Faculdade de Engenharia. Universidade do Porto. 2012

The role of vascular endothelial growth factor (VEGF) signaling in cancer is well-known in the context of angiogenesis but is also important in the functional regulation of tumor cells themselves. Notably, autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression and drug resistance. Therefore, the first half of this thesis focuses on the mechanism of VEGF-NRP-mediated support of CSCs. Aberrant activity of the Hippo pathway effector YAP and TAZ are associated with breast CSCs and have been shown to confer stem cell-like properties. I found that VEGF-NRP2 signaling contributes to the activation of YAP/TAZ in various breast cancer cells, which mediates a positive feedback loop that promotes mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, which enabled the activity of YAP/TAZ. In complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) β2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, YAP/TAZ-mediated repression of β2-chimaerin sustained Rac1 activity in CSCs. Depletion of β2-chimaerin in non-CSCs increased Rac1 activity, YAP/TAZ activation and mammosphere formation. Analysis of breast cancer patients revealed an inverse correlation between β2-chimaerin and TAZ expression in tumors. These findings highlight an unexpected role for β2-chimaerin in a feedforward loop of YAP/TAZ activation and the acquisition of CSC properties. Given that CSCs have been implicated in therapy resistance and are enriched in triple negative breast cancer (TNBC), which exhibits VEGF-NRP2 signaling, the second half of this thesis focuses on understanding the mechanism by which VEGF-NRP2 contributes to the chemoresistance of TNBC. I discovered that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double strand break repair. Mechanistically, I found that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. I also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to chemotherapy and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ in response to cisplatin. These findings reveal novel roles for VEGF-NRP2 and YAP/TAZ in DNA repair and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ and Rad51 that contributes to resistance to platinum chemotherapy. In summary, this thesis provides novel insight into the roles of autocrine VEGF-NRP2 signaling in breast CSC function and therapy resistance and provides rationale in inhibiting NRP2 for platinum-resistant tumors that are dependent on YAP/TAZ activation.

No description available.

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

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