A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Atypical teratoid rhabdoid tumor (ATRT) is a highly malignant pediatric central nervous system tumor. The prognosis is often poor, with a 2‐year survival rate estimated at 15%. This dismal prognosis highlights the need to develop new treatment modalities for this devastating pediatric tumor. Recently, a tumor suppressing signaling pathway known as Hippo has emerged as a possible cancer treatment target. The Hippo signaling pathway is involved in organ growth and maintenance, and is dysregulated in many diverse cancers. We used quantitative real‐time PCR to evaluate the mRNA expression profile of Hippo pathway genes. We then used determined the protein expression of various Hippo components using Western blots. The results of this study suggest that Hippo plays a definite role in atypical teratoid rhabdoid tumor.

Hippo signaling pathway represents organ size control mechanism constrained between all metazoans. Individual components of the Hippo signaling pathway were identified as key tumor-suppressors which phosphorylate and inhibit activity of several oncogenic factors and signaling pathways (such as YAP/TAZ, PI3K and mTOR). MST1 kinase is a part of central protein complex of the Hippo signaling pathway and its activation is involved in anti-cancer activity of several drugs. We have demonstrated activation of the MST1 kinase by natural compounds in leukemic cells followed by inhibition of proliferation and induction of apoptosis. Shikonin represents natural naphthoquinonic compound isolated from Lithospermum erythrorhizon which acts as inhibitor of glycolysis and mitochondrial respiratory chain in human cells. Shikonin induces fast activation of the MST1 protein in leukemic cells however mechanism of this activation remains unknown. Therefore, we tried to characterize posttranslational modifications of the MST1 kinase during shikonin treatment of leukemic cells. Firstly, we isolated MST1 kinase from control and shikonin-treated cells using immunoprecipitation. Then we characterized posttranslational modifications of the MST1 protein employing mass spectrometry. Using this approach we found out...

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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.

L’ovaire est un organe indispensable à la fonction reproductive, car il permet la production, la maturation et la libération de la cellule germinale femelle, l’ovocyte. Malgré son rôle central dans la régulation de la reproduction chez la femme, plusieurs de ses processus physiologiques et de ses conditions pathologiques sont encore imparfaitement décrits. La caractérisation du rôle de nouveaux régulateurs pourrait permettre l’élucidation de plusieurs questionnements actuels en physiologie ovarienne. Initialement étudiée dans l’organogenèse et l’oncogenèse pour son implication dans la prolifération, la migration, la différenciation et l’apoptose cellulaire, la voie de signalisation Hippo pourrait s’avérer être un facteur déterminant dans la physiologie ovarienne. En effet, elle a été récemment rapportée comme participant à la régulation de l’activation folliculaire, de la prolifération des cellules de la granulosa et de l’ovulation. La voie Hippo consiste en une cascade de kinases menant ultimement à la phosphorylation des deux co-régulateurs transcriptionnels YAP1 (yes-associated protein 1) et TAZ (transcriptional coactivator with a PDZ-binding motif). L’objectif général de ce projet de thèse était de caractériser les rôles de Yap1/Taz dans la physiologie ovarienne en utilisant des modèles murins transgéniques d’inactivation conditionnelle de ces gènes dans les cellules de la granulosa. La première portion du projet a conduit à la caractérisation d’un phénotype inattendu de défaut des oviductes. Les souris femelles adultes étaient sous-fertiles et leur fonction ovarienne était intacte. En fait, la sous-fertilité était causée par le piégeage des embryons dans des dilatations de la paroi de l’oviducte, empêchant ainsi leur transport adéquat vers l’utérus. Nous sommes parvenus à démontrer que la perte d’expression de YAP1/TAZ dans les couches musculeuses de l’oviducte conduisait à un amincissement progressif de sa paroi et était ultimement responsable de l’échec du transport embryonnaire. Dans la seconde portion du projet, nous avons utilisé la culture primaire de cellules de la granulosa afin de décrire l’implication de la voie Hippo dans l’ovulation. Nous avons identifié la protéine kinase A comme modulateur clé de l’activation de la voie Hippo par l’hormone lutéinisante (LH). En utilisant un système adénoviral de délétion de Yap1/Taz, nous avons mis en évidence l’importance de leur expression pour l’induction de plusieurs gènes cibles de la LH. Ensuite, au moyen d’une expérience d’immunoprécipitation de la chromatine, nous avons démontré l’implication de YAP1 dans la régulation de la transcription de l’amphiréguline, un effecteur central de la cascade de signalisation de la LH. Dans son ensemble, ce projet a permis de mettre la lumière sur de nouveaux rôles de la voie de signalisation Hippo dans la régulation des cellules musculaires lisses de l’oviducte et des cellules de la granulosa durant l’ovulation chez la souris. Elles ouvrent la voie à une investigation plus précise de l’implication de la voie Hippo dans ces deux organes clés du système reproducteur femelle. / The ovary is a central organ of the female reproductive tract involved in oocyte production, maturation and release. Still, many physiological and pathological ovarian processes remain to be described more comprehensively. The precise characterization of the roles of new ovarian regulators would contribute to a better understanding of its physiology. The Hippo signaling pathway was initially studied for its roles in cellular proliferation, apoptosis, migration and differentiation during organ and tumor development. Recently, it has been shown to be involved during normal physiological processes of multiple organs, including the ovary. Hippo was shown to be involved in the activation of primordial follicles, in the proliferation of granulosa cells and during ovulation. Hippo consists of a central kinase cascade leading to the phosphorylation of YAP1 (yes-associated protein 1) and TAZ (transcriptional coactivator with a PDZ-binding motif), the two transcriptional coactivators of the pathway. The objective of this thesis was to characterize the precise roles of Yap1/Taz in ovarian physiology using transgenic mouse models of their genetic deletion in granulosa cells. The first part of this thesis project led to the characterization of an unexpected oviductal phenotype. Adult females were subfertile and their ovarian function was unaffected. The subfertility was rather caused by the entrapment of embryos in oviductal dilations, preventing their normal transport to the uterus. We demonstrated that loss of YAP1/TAZ expression in oviductal smooth muscle cells led to a gradual thinning of the oviductal wall and was responsible for the embryo transport impediment. In the second part of this project, we cultured primary mouse granulosa cells to characterize the roles of the Hippo signaling pathway during ovulation. We showed that protein kinase A is a key effector of Hippo activation following the luteinizing hormone (LH) surge. We then demonstrated that Yap1/Taz expression is required for the induction of several LH target genes. Using a chromatin immunoprecipitation experiment, we were able to show that YAP1 drives the expression of amphiregulin, a key paracrine transmitter of the LH signal, during the early events of ovulation. Together, these results identified new roles of the Hippo signaling pathway in the regulation of oviductal smooth muscle cells and of granulosa cells during ovulation. This thesis project opens the door to new avenues of investigation of Hippo involvement in the regulation of the female reproductive system.