Dysregulated Hippo pathway signaling promotes the onset of aggressive cancers through the induced nuclear activity of yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) (YAP/TAZ). Uncontrolled nuclear YAP/TAZ activity evokes tumor-initiating properties in a range of epithelial-derived cancers, including oral and breast cancers, but their downstream targets and mechanisms of action are unclear. Recent studies have suggested that the pro-tumorigenic roles for YAP/TAZ relate to their convergence with growth factor signaling pathways. Based on these previous studies, I hypothesized that YAP/TAZ driven transcription contributes to carcinoma progression, and that cooperation with transforming growth factor β (TGFβ)-induced signals promotes aggressive oncogenic traits. In this thesis I show that dysregulated YAP localization precedes oral squamous cell carcinoma (OSCC) development, and that nuclear YAP/TAZ activity drives cell proliferation, survival, and migration in vitro, and is required for tumor growth and metastasis in vivo. Global gene expression studies in OSCC cells revealed that YAP/TAZ-mediated gene expression correlates with expression changes that occur in human OSCCs identified by “The Cancer Genome Atlas” (TCGA), many of which encode cell cycle and survival regulators. By exploring the relationship with growth factor signaling, I found that YAP/TAZ induce pro-tumorigenic events by converging with TGFβ-induced signals, particularly in breast cancer cells where TGFβ is known to promote metastatic properties. My observations indicated that YAP/TAZ are necessary for maintaining and promoting TGFβ-induced tumorigenic phenotypes in breast cancer cells, and that these phenotypes result from the cooperative activity of YAP/TAZ, the TEA domain family of transcription factors (TEADs), and TGFβ-activated SMAD2/3 in the nucleus. Genome-wide expression analyses indicated that YAP/TAZ, TEADs, and TGFβ-induced signals coordinate a specific pro-tumorigenic transcriptional program. Importantly, genes cooperatively regulated by these complexes, such as the novel targets neuronal growth regulator 1 (NEGR1) and urothelial cancer associated 1 (UCA1), are necessary to maintain tumorigenic activity in metastatic breast cancer cells. Nuclear YAP/TAZ also cooperate with TGFβ signaling to promote phenotypic and transcriptional changes in non-tumorigenic cells to overcome TGFβ-mediated growth inhibition. This work thus defines novel roles for YAP/TAZ in cancer, offering molecular mechanisms that may be useful for identifying and targeting YAP/TAZ-driven cancers.

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.

TAZ (Transcriptional co-activator with PDZ-binding motif) is a WW-domain containing protein recently identified as a downstream component of the Hippo tumor suppressor pathway and mediator of biologically important processes (mesenchymal stem cell differentiation, embryonic stem cell renewal, mechanotransduction). Recently, loss of LATS1/2, a negative regulator of TAZ, has been observed in ~50% of breast cancers. However, whether and how TAZ is also involved in breast cancer has not been investigated. Therefore, this study explores the cellular functions of TAZ in breast cancer and the underlying molecular mechanisms. The cellular functions of TAZ were investigated using overexpression studies in an immortalized mammary epithelial cell line (MCF10A). Compared to control vector-only expressing cells (MCF10A-WPI) TAZ overexpression (MCF10A-TAZ) promotes enhanced cell proliferation, cell migration and cell-ECM adhesion, induces transformation and the epithelial-mesenchymal transition, and confers resistance to chemotherapeutics (paclitaxel, cisplatin). Together, these findings strongly suggest TAZ functions as an oncogene in the development, progression and drug resistance of breast cancer. As a transcriptional co-activator, TAZ likely mediates these cellular functions through the transcriptional activation of downstream genes. By screening a 44K human genome microarray we have identified and characterized Cyr61 and CTGF, mediators of paclitaxel resistance, and BMP4, a regulator of cell migration. Through stable shRNA-mediated knockdown, we show that loss of Cyr61/CTGF expression in MCF10A-TAZ cells can rescue TAZ-induced paclitaxel resistance. Similarly, shRNA-mediated knockdown of BMP4 can significantly attenuate TAZ-induced cell migration. Therefore, these findings demonstrate that Cyr61/CTGF and BMP4 are functionally significant mediators of TAZ-induced paclitaxel resistance and cell migration, respectively. The clinical relevance of our in vitro findings were also validated by immunohistochemistry using tissue microarrays containing human breast cancer samples. TAZ levels were highly expressed in 66.6% of clinical samples further suggesting TAZ may be an important oncogene in breast cancer. Our study has characterized TAZ as an oncogene in breast cancer and elucidated two novel mechanisms underlying paclitaxel resistance and cell migration. These findings highlight the importance of TAZ during the development, progression and drug resistance of breast cancers and the potential use of TAZ as a therapeutic target to treat TAZ-expressing breast cancers. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-08-01 23:59:08.68

We have previously demonstrated that Llgl1 loss results in a gain of mesenchymal phenotypes and a loss of apicobasal and planar polarity. We now demonstrate that these changes represent a fundamental shift in cellular phenotype. Llgl1 regulates the expression of multiple cell identity markers, including CD44, CD49f, and CD24, and the nuclear translocation of TAZ and Slug. Cells lacking Llgl1 form mammospheres, where survival and transplantability is dependent upon the Epidermal Growth Factor Receptor (EGFR). Additionally, Llgl1 loss allows cells to grow in soft-agar and maintain prolonged survival as orthotopic transplants in NOD-SCID mice. Lineage tracing and wound healing experiments demonstrate that mammosphere survival is due to enhanced EGF-dependent migration. The loss of Llgl1 drives EGFR mislocalization and an EGFR mislocalization point mutation (P667A) drives these same phenotypes, including activation of AKT and TAZ nuclear translocation. Together, these data indicate that the loss of Llgl1 results in EGFR mislocalization, promoting pre-neoplastic changes.

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.

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.

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

The Hippo tumor suppressor pathway is a highly conserved signaling pathway that regulates cell proliferation, differentiation, and organ size. Activation of the Hippo pathway leads to the phosphorylation and cytoplasmic sequestration of the pro-growth transcriptional co-activators YAP/TAZ; by contrast, impairment of the Hippo pathway enables YAP/TAZ to enter the nucleus where they bind to the TEAD transcription factors and induce the expression of genes involved in cell proliferation. Functional impairment of the Hippo pathway, and subsequent hyperactivation of YAP/TAZ, is common in many human malignancies, including melanoma. Recently, small molecule inhibitors that disrupt YAP/TAZ-TEAD binding, and thus reduce oncogenic transcriptional signaling have been discovered, but their efficacy in preventing cancer cell growth has not yet been well characterized. Moreover, no simple biomarker has been identified that can predict sensitivity to such inhibitors. We hypothesized that cells in which YAP/TAZ are enriched in the nucleus relative to the cytoplasm, indicative of an impaired Hippo pathway, would be more susceptible to TEAD inhibition. This would provide a useful biomarker to identify cancer cell lines most likely to respond to TEAD inhibition. We therefore developed and validated an automated quantification method to score nuclear:cytoplasmic YAP/TAZ localization in melanoma cell lines. This enabled us to identify “Hippo-On” and “Hippo-Off” signatures. We then treated these lines with the TEAD inhibitor MGH-CP1 and performed cell viability assays. Results from these data demonstrated that cell lines that have greater nuclear localization of YAP/TAZ are more susceptible to MGH-CP1 inhibition, suggesting that YAP/TAZ nuclear localization may be a biomarker to identify candidates for TEAD inhibitor treatment.