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

by Chan Wood-yee. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1987. / Includes bibliographical references.

Accumulating evidence highlights the importance of studying the migration of cells within the context of their natural environment as manipulating the substrate on which a cell is migrating can have a dramatic impact on the mode/mechanisms employed by cells during migration. Central to this phenomenon is the requirement of adhesion to the ECM in order to gain traction during migration. Integrins constitute the main family of cell receptors involved in mediating cell-ECM interactions during motility. Whilst traditionally two-dimensional cell culture studies have placed emphasis on the importance of these receptors for spreading and migration, it has become evident that within more confined environments these receptors, at least for some cell types, are less crucial. In this research we utilise Drosophila embryonic hemocytes as an in vivo model for cell migration. We show that whilst hemocytes migrate within confined environments in vivo, these cells depend on integrins for powering both developmental and inflammatory migrations. Given the close association between these receptors and the actin cytoskeleton we were surprised to discover that removal of the main β integrin subunit, Myospheroid, did not affect cell spreading in vivo and had only a small impact on lamellipodial structure and dynamics. Furthermore we discovered that, in contrast to other cell types previously analysed, removal of this integrin subunit in hemocytes was not accompanied by an increase in the rate of actin retrograde flow within the protrusions, which we believe could reflect abrogation of a positive feedback between Rho, ROCK and Myosin II contraction. Instead, we discover a key role for integrins in regulating the microtubule cytoskeleton, in the maintenance of a polarised microtubule bundle, termed a ‘microtubule-arm’. Although the molecular mechanisms by which this stabilisation is coordinated have yet to be identified, this provides important insight into the co-regulation of adhesion and microtubule cytoskeleton important for the migratory behaviour of these cells. Cell migration reflects the complex and integrated regulation of the actin cytoskeleton by diverse families of actin regulatory proteins. Using hemocytes as a model system, we also explore the regulatory interactions between two main actin regulatory proteins, Diaphanous and Enabled, in vivo. Whilst the function of these proteins in the formation of filopodial protrusions is overlapping, recent research has highlighted the ability of these proteins to regulate the activity of one another. We find that co-expression of Enabled in hemocytes is able to rescue the morphological and migratory defects resulting from overexpression of active Diaphanous. Thus, data here presents Enabled as a negative regulator of Diaphanous, which may play an important role in the migration of hemocytes in vivo.

Cell competition is a form of cell-cell signalling that results in the elimination of less fit cells from a tissue by their fitter counterparts. I take advantage of an established in vitro model of cell competition using Madin-Darby canine kidney (MDCK) cells to shed insight into the molecular basis of cell competition in epithelial cells. In this system, silencing of the tumour suppressor scribble (scribKD) results in a 'loser' phenotype whereby scribKD cells are specifically eliminated from the monolayer by surrounding wild-type cells. More specifically, scribKD cells are compacted into tight clones through activation of a directed, collective migration in the wild-type population: scribKD are 'mechanical losers' and delaminate and die due to an intrinsic hypersensitivity to high cell density. Remarkably, p53 activation is both necessary and sufficient for this mechanical loser cell status. I first investigate the role of E-, N-, and P-cadherin in the directed migration between scribKD and wild-type cells and in scribKD cell loser status. I show that differential expression of E-cadherin between scribKD losers and wild-type winners is required but not sufficient for directed migration and has no impact on loser cell status. I also show that elevation of neither E-cadherin nor N-cadherin is sufficient to induce directed migration or loser status, but that P-cadherin may play a role in both. I next focus on translating findings about the molecular details of competition from the scribKD set-up into a system where p53 differences alone drive the formation and elimination of mechanical losers. I show that the ROCK - P-p38 - p53 pathway activated in response to mechanical compaction in scribKD cells is conserved in p53-driven losers. In the latter part of my thesis, I characterise the directed migration observed during MDCK competition by drawing parallels to canonical leader-follower migration. Canonical leader cells emerge when epithelial sheets are wounded and, by becoming migratory, drive collective cell migration of follower cells, which results in wound closure. It was not known what confers the leader cell fate. I show that p53 and its effector p21 (and potentially other cyclin-dependent kinase inhibitors) are the key drivers of leader cell migration. I demonstrate that p53-induced leaders use the same molecular pathways that have been shown to drive leader cell migration during wound healing and, in fact, p53 and p21 are also elevated in leaders generated by wounding. Importantly, I establish that p53 activity drives efficient wound closure. Lastly, I show that leader cells are often eliminated by cell competition in the final stages of wound closure, as their elevated p53 mediates their hypersensitivity to density. The model incorporating these data proposes that cellular damage during wounding generates cells with elevated p53, which become leaders and drive wound healing, but these are then cleared once the wound is closed because their high p53 levels cause them to become mechanical losers.

O carcinoma espinocelular é uma neoplasia maligna que representa aproximadamente 94% de todas as ocorrências presentes em boca e uma das suas principais características celulares é a migração de suas células para formar metástases. A adesão celular é considerada um dos eventos determinantes da migração celular. Para as células formarem uma estrutura tecidual tridimensional as adesões entre células e entre células e matriz extracelular são de grande importância. As junções de adesão celulares surgem, caracteristicamente, pela interação entre receptores adesivos, vias de sinalização e elementos do citoesqueleto. A proteína E-caderina está presente em adesões entre células no tecido epitelial. A proteína FAK está envolvida na maioria dos eventos relacionados à adesão celular estimulada por integrinas. A Vinculina é uma proteína de adesão que se liga ao citoesqueleto de actinomiosina como uma proteína de adesão focal através das integrinas. Estudos recentes sugerem que há alteração na expressão e atividade de proteínas de adesão em tumores malignos. O objetivo deste trabalho foi descrever o padrão de expressão e de regulação da atividade de proteínas de adesão em amostras de tumores de carcinoma espinocelular. Foram realizadas reações de imunoistoquímica para verificar o padrão de distribuição das proteínas E-caderina, Vimentina e FAK-y397 em amostras de tumores de carcinoma espinocelular oral. Verificou-se a diminuição da expressão de E-caderina e de Vinculina em regiões de adesão célula-célula e em contrapartida constatou-se aumento na marcação citoplasmática de Vinculina bem como na marcação de FAK-y397 em todas as amostras de tumores. Apesar dos avanços, ainda são necessários mais estudos observacionais que averiguem não apenas o grau de expressão dessas proteínas de adesão, mas também o seu nível de regulação. A partir dos resultados deste estudo, pode-se sugerir que o controle do nível de expressão e de atividade da adesão celular podem ser considerados como potenciais alvos para a aplicação de terapias coadjuvantes que visam a diminuir ou impedir a progressão tumoral, bem como o desenvolvimento de metástases. / Squamous cell carcinoma is a malignant neoplasm that accounts for approximately 94% of all occurrences present in mouth and one of its main characteristics is the cellular migration of its cells to form metastases. Cell adhesion is considered one of the defining events of cell migration. For a three-dimensional tissue structure, adhesions between cells and between cells and the extracellular matrix is of great importance. Cell adhesion junctions arise characteristically by interaction between adhesive receptors, signaling pathways and cytoskeletal elements. The protein E-cadherin is present in cells in the adhesion between epithelial tissue. The Focal Adhesion Kinase (FAK) protein is involved in most events related to cell adhesion stimulated by integrins. The vinculin is an adhesion protein that binds cytoskeletal protein through integrins activaion. Recent studies suggest that there are alterations in the expression and activity of adhesion proteins in malignant tumors. The aim of this study was to describe the pattern of expression and regulation of the activity of adhesion proteins in tumor samples of squamous cell carcinoma. Immunohistochemical reactions were performed to check the distribution pattern of the protein E-cadherin, vimentin and FAK-y397 in tumor samples of oral squamous cell carcinoma. There was a decrease in the expression of E-cadherin and vinculin in regions of cell-cell adhesion but, on the other hand, it was found to increase in cytoplasmic as well as unscheduled vinculin FAK-y397 in all tumor samples. Despite progress, it is necessary more observational studies that examine not only the degree of expression of these adhesion proteins, but also its level of regulation. From the results of this study it is suggested that the control of the expression level and activity of cell adhesion may be considered as potential targets for application adjuvant therapies that aim to reduce or prevent tumor progression and the development metastases.

The I-BAR proteins are a family of actin regulators which include IRSp53 and Missing-In-Metastasis (MIM). These proteins possess an N-terminal I-BAR domain which associates with both the actin cytoskeleton and membrane phospholipids and is able to induce membrane curvature. Previous cell culture and in vitro studies have implicated I-BAR proteins in the regulation of actin protrusion formation, however their roles within the organism are poorly understood. We have used Drosophila melanogaster as a model system in which to study I-BAR protein function at the cellular and organismal level. Drosophila possess two I-BAR proteins, one homologous to IRSp53 and one homologous to MIM. Using full- length and truncated splice variants generated for both dMIM and dIRSp53, we have performed structure/function analysis to determine the role of specific domains in the localisation and actin modifying abilities of the proteins in both cell culture and in vivo. We found that dMIM overexpression typically promotes a lamellipodial morphology, with dMIM localising to the edges of extending lamellipodia. dIRSp53 expression induced a more filopodial phenotype in cell culture, which was not as notable in vivo, however expression of a dIRSp53 splice variant with a WH2 domain resulted instead in a predominantly lamellipodial morphology. Similar to dMIM, dIRSp53 localises to the tip of extending protrusions, albeit more transiently. We found that multiple domains contribute to the localisation and activity of dIRSp53 and dMIM. Following overexpression analysis, complementary loss-of-function analysis was performed in vivo using Drosophila mutants lacking dMIM, dIRSp53 or both genes together. Surprisingly these mutants were viable and morphologically normal. Absence of these genes individually or together did not greatly affect cell migration or actin dynamics in haemocytes or epithelial cells undergoing dorsal closure. However, a role for I- BAR proteins in axonal filopodia formation within primary neuronal cultures was apparent, as was a notable role in neuromuscular junction morphology. We have also identified potential redundancy between Drosophila MIM and the Drosophila F-BAR protein Cip4 in actin bundle regulation within embryonic haemocytes, with an additional novel role for Cip4 alone in haemocyte lamellipodial maintenance. Our results suggest that the Drosophila I-BAR proteins contribute to actin cytoskeleton regulation in vitro and in vivo, particularly within the CNS, and with novel shared functions with other BAR domain family proteins contributing to their regulation of actin cytoskeletal organisation and function.

Glucocorticoids (GCs) are a class of steroid hormone that play essential roles in development, glucose homeostasis, and reducing inflammation. Clinically, GCs are potent anti-inflammatory and immunosuppressive agents used to treat a variety of diseases. However, the therapeutic benefit of GCs is negatively impacted by the induction of severe side effects. In this thesis, I present two studies that have contributed to the understanding of the non-genomic actions of GCs. GCs inhibit cell migration by a non-transcriptional pathway involving HDAC6: A negative side effect of GC therapy is impaired wound healing which is ascribed to inhibited cell migration. Using live-cell microscopy, I show that GCs inhibit cell migration within 30 minutes of administration. GCs alter the dynamics of the microtubule network through rapid induction of tubulin acetylation (by inhibition of HDAC6) which increases microtubule stability and slows cell movement. The inhibitory effect of GCs on cell migration is reversed by overexpressing HDAC6. Using quantitative imaging, I identified a rapid ligand-dependent association of the GR and HDAC6 within the cytoplasm that is absent in the nucleus. However, a very small proportion of HDAC6 enters the nucleus post-GC treatment, suggesting that HDAC6 accompanies the GR during nuclear translocation. This study demonstrates that GCs rapidly inhibit cell migration by a non-transcriptional mechanism involving HDAC6. Investigating the rapid effects of GCs on the phosphoproteome: Non-steroidal GCs are useful tool compounds to dissect glucocorticoid receptor (GR) activity. Here, I investigated the early, rapid effect of GCs on the phosphoproteome of A549 cells using SILAC-based phosphoproteomics. A consistent spectrum of phosphoproteins was differentially regulated by GC within 10 minutes of administration, notably including regulators of RNA polymerase II, chromatin remodifying proteins, transcription factors, cytoskeletal modifiers, regulators of intracellular calcium signalling and endocytosis. These phosphoproteins were validated by western blotting. This study shows a clear early effect of GCs on the phosphoproteome with implications for non-specific, non-transcriptional activity of GCs.

In this thesis I explore how migratory properties of the model organism Dictyostelium discoideum are influenced by dimensionality and topology of the environment that surrounds the cell. Additionally, I sought to develop a microfluidic device able to measure mechanical properties of single cells with a sufficient throughput to account for the inherent heterogeneity of biological samples. Throughout this thesis I made use of microfabrication methods such as photo-lithography and soft-lithography, to develop ad hoc microstructured substrates. These tools enabled me to tackle different biological and biomedical questions related to cell migration and cell mechanics. Confining cells into channels with low dimensionality appeared to regulate the velocity of cellular locomotion, as well as the migration strategy adopted by the cell. Spatial confinement induced an altered arrangement of the acto-myosin cytoskeleton and microtubules. Moreover, the spatial constraint resulted in a simplified, mono-dimensional migration, characterised by constant average speed. Additionally, some cellular processes tended to occur in a periodic fashion, upon confinement. Interestingly, if Dictyostelium cells migrated through asymmetric bifurcating micro- channels, they appeared to be able to undergo a ’decision-making’ process leading to a directional bias. Although the biophysical mechanism underlying this response is yet to be understood, the data shown in this thesis suggest that Dictyostelium cells respond to differences in local concentrations of chemoattractants. The speed of a cell that crawls in a channel also depends on the cell’s stiffness, that in turn represents a measure of the density and structure of its cytoskeleton. To date, only a few methods have been developed to investigate cell mechanics with sufficient throughput. This motivated my interest in developing a microfluidic-based device that, exploiting the recording capabilities of a modern high speed camera, enabled me to assess the cellular mechanical properties at a rate greater than 10,000 cells per second, without the need for cell labelling. In this thesis I presented an example of how this method can be employed to detect differences between healthy and cancerous prostate cells, as well as to differentiate between prostate and bladder cancer cells based on their mechanical response. In conclusion, the work presented in this thesis highlights the interdisciplinarity required to investigate complex biological and biomedical problems. Specifically, the use of quantitative approaches that span from microtechnology, live imaging, computer vision and computational modelling enabled me to investigate novel biological processes as well as to explore new diagnostic technologies that aim to promote the improvement of the future healthcare.

Copy of author's previously published work inserted. Bibliography: leaves 139-160.

Copy of author's previously published work inserted. / Bibliography: leaves 139-160. / 160, [10] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001?