Oncogenes drive cancer by hijacking normal cellular functions involved in proliferation and survival. Suppression of the driving oncogene is highly effective for promoting tumor regression, a phenomenon termed "oncogenic addiction." By using unbiased genetic tools to functionally probe oncogenic addiction, we can identify cancer dependencies and characterize aspects of oncogenic signaling.

Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 60,000 annual diagnoses in the U.S. and approximately 500,000 worldwide. About 90% of these individuals receive radiation therapy, and salivary hypofunction and xerostomia occur in 60-85% of these patients due to irreversible damage to the salivary glands. Current preventative and palliative care fail to improve quality of life, accentuating the need for regenerative therapies. Stem/progenitor-cell based therapies have been proposed to regenerate the irradiated glands; however, the identity of stem and progenitor cells in the adult salivary glands has remained somewhat elusive. Moreover, it is unclear how salivary progenitors respond to radiation and whether they can be stimulated to effectively reinstate salivary function. The second chapter of the present study describes the development of a label-retaining assay in salivary glands using EdU. The label-retaining cells (LRCs) identified in murine salivary glands have proliferative potential in vitro and expressed markers of putative salivary progenitors, such as Keratin 5, Keratin 14, and c-Kit. Interestingly, LRCs were still present 30 days following radiation, when chronic loss of saliva is evident. The significance of these findings lies in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining salivary gland homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. In the following chapter, we show that a unique population of murine salivary gland LRCs undergo compensatory proliferation in response to radiation. The initiation of compensatory proliferation is tightly associated with inactivation of the kinase aPKCζ and increased nuclear localization of YAP. This part of the study provides novel insights into the regulation of function of salivary gland progenitors, which can be utilized for the development of therapeutic agents to treat salivary hypofunction. Finally, the last chapter describes how the mechanisms found to initiate compensatory proliferation in acinar LRCs as a response to radiation are involved in the regeneration of salivary glands with IGF-1. Administration of IGF-1 post-radiation restores salivary function in mice, but the mechanisms of regeneration are still unknown. Here, we show that IGF-1 requires aPKCζ to restore saliva production. Further, IGF-1 inhibits nuclear translocation of Yap in an aPKCζ-dependent fashion. We propose that a tightly regulated balance in the levels of aPKCζ and Yap in acinar LRCs has to be maintained in order to restore function following radiation. In conclusion, the findings from this study provide new knowledge in regards to the regulation of function of salivary progenitors during a state of injury (by radiation) and during regeneration (with IGF), and offer potential targets of study for the development of new therapeutics for salivary gland dysfunction. Future studies will determine whether aPKCζ and Yap can be effectively targeted in salivary progenitors to restore salivary function in head and neck cancer patients who receive radiation therapy.

Lung cancer is the leading cause of cancer deaths worldwide. A large part of this high mortality rate is due to the onset of metastatic disease prior to diagnosis. Advances in treatment for metastatic disease may be achieved by understanding more about the identity of metastatic tumor cells and the mechanisms those cells employ to spread throughout the body. This thesis examined the relationship between cells capable of tumor propagation upon serial transplantation (tumor-propagating cells, or TPCs) and those with metastatic potential.

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.

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.

Elucidating the molecular events that integrate the patterning, morphogenesis, and differentiation of epithelial progenitor cells into complex tissues is a primary focus of epithelial developmental biology research. Expansion and maintenance of epithelial progenitor populations is crucial for developmental events, but growth must be tightly coupled to consequent cellular differentiation and specialization. The Hippo pathway has surfaced as an important regulator of epithelial progenitor identity: nuclear activity of the Hippo effector Yap maintains epithelial progenitor status while Hippo-mediated nuclear exclusion of Yap by the Lats1/2 kinases induces differentiation. Extending this general theme into an additional organ system, the submandibular gland (SMG), as well as identifying upstream regulators of Yap and Lats1/2 in the developing lung was the goal of this work. Here, we describe important roles for Yap in the morphogenesis and patterning of lung and SMG epithelium, both of which are composed of highly organized branched structures. Epithelial-specific genetic ablation of Yap as well as its upstream negative regulators Lats1/2 was used to interrogate loss- and gain-of-function phenotypes, whereby Lats1/2 ablation is known to result in unrestricted nuclear Yap activity. Loss of Yap in the SMG resulted in a striking deficiency of Krt5/Krt14-positive epithelial progenitor populations accompanied by impaired branching morphogenesis. Deletion of Lats1/2 in the SMG resulted in a massive expansion of Krt5/Krt14-positive epithelial progenitor populations that failed to terminally differentiate. As epithelial progenitors in the lung and SMG begin to differentiate, they also acquire distinct morphologies. In both the lung and the SMG, Krt5-positive basal cells lie beneath a layer of Krt8/Krt19-positive luminal cells. We observed that luminal cells exhibited a columnar morphology while basal cells retained a cuboidal morphology, and that this difference correlated with the expression of the polarity protein Crb3. After ablating Crb3 in the developing lung epithelium, luminal cells were unable to polarize, exhibited aberrant nuclear Yap activity, and remained in a progenitor state. Crb3 functions to initiate Lats1/2 activity, promoting Yap phosphorylation and its consequent nuclear exclusion, which drives differentiation. Taken together, this work identifies essential roles for polarity/Hippo pathway-mediated control of Yap activity in epithelial progenitor expansion and differentiation. / 2018-07-09T00:00:00Z

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

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.

京都大学 / 新制・論文博士 / 博士(生命科学) / 乙第13431号 / 論生博第26号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 石川 冬木, 教授 原田 浩 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Proper lung function relies on the precise balance of specialized epithelial cell types that coordinate to maintain homeostasis. The Hippo pathway has emerged as a critical regulator of cell fate both developmentally and in a regenerative setting. The work presented in this dissertation describes essential roles for the transcriptional effectors of Hippo pathway signaling, Yap and Taz, in maintaining lung epithelial homeostasis. The data presented here demonstrate that conditional deletion of Yap and Wwtr1/Taz in the lung epithelium of adult mice results in severe defects with consequent animal lethality. Phenotypes associated with Yap/Taz deletion include alveolar disorganization, a development of mucin hypersecretion throughout the airways, and ciliary disorganization. Through in vivo lineage tracing, analysis of mouse and human tissues, along with in vitro molecular experiments, these studies show that nuclear YAP/TAZ exert transcriptional control over club cell fate, while in multiciliated cells, they function within the cytoplasm to maintain ciliary structures. Within club cells, reduced YAP/TAZ activity promotes intrinsic goblet transdifferentiation of secretory airway epithelial cells. Global gene expression and ChIP-Seq analyses reveal that YAP/TAZ act through the TEAD family of transcription factors to suppress a goblet cell differentiation program in airway epithelial cells, including direct repression of the SPDEF gene, which encodes a transcription factor required for goblet cell identity. Further in vitro studies identify cooperation between YAP/TAZ-TEAD and the NuRD chromatin remodeling complex to inhibit SPDEF expression and that Hippo-regulated YAP/TAZ impinge on cytokine-induced goblet cell differentiation. Within multiciliated cells, we observe that phosphorylated Yap localizes in a planar polarized manner at the base of cilia and controls ciliary and basal body density. Lineage specific Yap/Taz deletion leads to reduced ciliary density and height due to a loss of apically basal bodies. Collectively, this work identifies YAP/TAZ as critical factors in lung epithelial homeostasis and offers new molecular insight into the mechanisms regulating the secretory and multiciliated cell lineages, which are frequently impaired in a broad range of lung diseases. / 2024-02-02T00:00:00Z