Dissertations / Theses on the topic 'Epithelial-mesenchymal transition'

Oral squamous cell carcinoma (OSCC), derived from normal oral epithelium transformation, remains a major public health problem world-wide. The prognosis of OSCCs that occur on lips is good, while other sites of oral mucosa where OSCC appears are more progressive, invasive and metastatic. A small subset of cells within a malignant neoplasm, named cancer stem cells (CSCs) or tumour initiating cells are thought to be capable of initiating the neoplasm itself, and of driving its growth and recurrance after treatment. The precise origin of CSCs is an ambiguous issue at present. The first proposal of the origin of CSCs is that CSCs develop from tumour cells themselves via cellular dedifferentiation. The secondary hypothesis for the origin of CSCs proposes that CSCs are the product of malignant transformation of adult stem cells. In this Ph.D thesis, we tried to demonstrate that CSCs in OSCC may be produced from those pathways.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
Griffith Health
Full Text

Although heterochromatin is enriched with repressive traits, it is actively transcribed, giving rise to large amounts of non-coding RNAs. These transcripts are responsible for the formation and maintenance of heterochromatin, but little is known about how their transcription is regulated. In this thesis we show that Snail1 transcription factor represses mouse pericentromeric transcription and regulates heterochromatin organization through the action of the H3K4 deaminase LOXL2. Snail1 has a key role in epithelial-to-mesenchymal transition (EMT). We show that, also during this process, Snail1 is responsible for pericentromeric transcription regulation. At the onset of EMT, one of the major structural heterochromatin proteins, HP1α, is transiently released from heterochromatin foci in a Snail1/LOXL2 dependent manner, concomitantly with a down-regulation of major satellite transcription. Moreover, prevention of major satellite transcripts down-regulation compromises the migratory and invasive behaviour of EMT resulting mesenchymal cells. We propose that Snail1 and LOXL2 regulate heterochromatin during this process, which may be crucial to allow the genome reorganization required to complete EMT.
Tot i estar enriquida en marques repressores, l’heterocromatina es transcriu activament i dóna lloc a grans quantitats d’ARNs no codificants. Aquests trànscrits són responsables de la formació i el manteniment de l’heterocromatina, però com es regula la seva transcripció segueix sent quelcom poc clarificat. En aquesta tesi demostrem que el factor de transcripció Snail1 reprimeix la transcripció pericentromèrica en cèl·lules de ratolí i regula l’organització de l’heterocromatina a través de l’acció de la LOXL2, que deamina l’H3K4. Snail1 té un paper clau en la transició epiteli-mesènquima (EMT). Aquí demostrem que, també durant aquest procés, Snail1 és responsable de la regulació de la transcripció pericentromèrica. A l’inici de l’EMT, l’HP1α, una de les principals proteïnes estructurals de l’heterocromatina, es desprèn de forma transitòria de l’heterocromatina. Aquest esdeveniment està regulat per Snail1 i LOXL2 i coincideix amb una disminució de la transcripció pericentromèrica. El bloqueig de la baixada dels trànscrits durant l’EMT compromet les capacitats migratòries i invasives de les cèl·lules mesenchimals que en resulten. Així doncs, proposem que Snail1 i LOXL2 regulen l’heterocromatina durant aquest procés, i així permeten que tingui lloc la reorganització genòmica que deu ser necessària per tal que es completi la EMT.

The transforming growth factor beta (TGFβ) is a cytokine that regulates a plethora of cellular processes such as cell proliferation, differentiation, migration and apoptosis. TGFβ signals via serine/threonine kinase receptors and activates the Smads to regulate gene expression. Enigmatically, TGFβ has a dichotomous role as a tumor suppressor and a tumor promoter in cancer. At early stages of tumorigenesis, TGFβ acts as a tumor suppressor by exerting growth inhibitory effects and inducing apoptosis. However, at advanced stages, TGFβ contributes to tumor malignancy by promoting invasion and metastasis. The pro-tumorigenic TGFβ potently triggers an embryonic program known as epithelial-mesenchymal transition (EMT). EMT is a dynamic process whereby polarized epithelial cells adapt a mesenchymal morphology, thereby facilitating migration and invasion. Downregulation of cell-cell adhesion molecules, such as E-cadherin and ZO-1, is an eminent feature of EMT. TGFβ induces EMT by upregulating a non-histone chromatin factor, high mobility group A2 (HMGA2). This thesis focuses on elucidating the molecular mechanisms by which HMGA2 elicits EMT. We found that HMGA2 regulates a network of EMT transcription factors (EMT-TFs), such as members of the Snail, ZEB and Twist families, during TGFβ-induced EMT. HMGA2 can interact with Smad complexes to synergistically induce Snail expression. HMGA2 also directly binds and activates the Twist promoter. We used mouse mammary epithelial cells overexpressing HMGA2, which are mesenchymal in morphology and highly invasive, as a constitutive EMT model. Snail and Twist have complementary roles in HMGA2-mesenchymal cells during EMT, and tight junctions were restored upon silencing of both Snail and Twist in these cells. Finally, we also demonstrate that HMGA2 can epigenetically silence the E-cadherin gene. In summary, HMGA2 modulates multiple reprogramming events to promote EMT and invasion.

Epithelial to Mesenchymal Transition (EMT) is a cellular transformation process that is employed repeatedly and ubiquitously during vertebrate morphogenesis to build complex tissues and organs. Cellular transformations that occur during cancer cell invasion are phenotypically similar to developmental EMT, and involve the same molecular signalling pathways. EMT processes are diverse, but are characterised by: a loss of cell-cell adhesion; a gain in cell-matrix adhesion; an increase in cell motility; the secretion of proteases that degrade basement membrane proteins; an increased resistance to apoptosis; a loss of polarisation; increased production of extracellular matrix components; a change from a rounded to a fibroblastic morphology; and an invasive phenotype. This thesis focuses explicitly on endocardial EMT, which is the EMT that occurs during vertebrate embryonic heart development. The embryonic heart initially forms as a tube, with myocardium externally, endocardium internally, with these tissue layers separated by a thick extracellular matrix termed the cardiac jelly. Some of the endocardial cells in specific regions of the embryonic heart tube undergo EMT and invade the cardiac jelly. This causes cellularised swellings inside the embryonic heart tube termed the endocardial cushions. The emergence of the four chambered double pump heart of mammals involves a complex remodelling that the endocardial cushions play an active role in. Even while heart remodelling is taking place, the heart tube is operating as a single-circulation pump, and the endocardial cushions are performing a valve-like function that is critical to the survival of the embryo (Nomura-Kitabayashi et al. 2009). As the endocardial cushions grow and remodel, they become the valve leaflets of the foetal heart. The endocardial cushions also contribute tissue to the septa (walls) of the heart. Their correct formation is thus essential to the development of a fully functional, fully divided, double-pump system. It has been shown that genetic mutations that cause impaired endocardial EMT lead to the development of a range of congenital heart defects (Fischer et al. 2007). An extensive review is conducted of existing experimental investigations into endocardial EMT. The information extracted from this review is used to develop a multiscale conceptual model of endocardial EMT, including the major protein signalling pathways involved, and the cellular phenotypes that they induce or inhibit. After considering the requirements for computational simulations of EMT, and reviewing the various techniques and simulation packages available for multi-cell modelling, cellular Potts modelling is selected as having the most appropriate combination of features. The open source simulation platform Compucell3D is selected for model development, due to the flexibility, range of features provided and an existing implementation of multiscale models; that include subcellular models of reaction pathways. Based on the conceptual model of endocardial EMT, abstract computational simulations of key aspects are developed, in order to investigate qualitative behaviour under different simulated conditions. The abstract simulations include a 2D multiscale model of Notch signalling lateral induction, which is the mechanism by which the embryonic heart tube is patterned into cushion and non-cushion forming regions. Additionally, a 3D simulation is used to investigate the possible role of contact-inhibited mitosis, upregulated by the VEGF protein, in maintaining an epithelial phenotype. One particular in vitro investigation of endocardial EMT (Luna-Zurita et al. 2010) is used to develop quantitative simulations. The quantitative data used for fitting the simulations consist of cell shape metrics that are derived from simple processing of the imaging results. Single cell simulations are used to investigate the relationship between cell motility and cell shape in the cellular Potts model. The findings are then implemented in multi-cell models, in order to investigate the relationship between cell-cell adhesion, cell-matrix adhesion, cell motility and cell shape during EMT.

Epithelial-mesenchymal transition (EMT) is a fundamental developmental program, which is believed to be reactivated during the progression of in situ carcinoma to aggressive metastatic cancers. Ras-ERK pathway has been shown to play a crucial role in EMT. We have previously shown that ERK2, but not ERK1, is necessary for RasV12-induced EMT and overexpression of ERK2 is sufficient to promote EMT. ERK2 promotes EMT by regulating several factors, including the upregulation of transcription factors ZEB1/2. ZEB1/2 repress expression of E-cadherin, which is necessary for polar epithelial tissue formations.

Background and aims: Increasing evidence suggests that epithelial to mesenchymal transition (EMT) has a key role in breast cancer progression, underlying invasion, metastatic dissemination and acquisition of therapeutic resistance. However, this role is predominantly inferred from in vitro and animal studies and controversy regarding EMT in human cancer remains. This thesis has two principle aims. Firstly, to clarify the role of EMT in human breast cancer at the protein level. Secondly, to develop a three-dimensional in vitro assay to investigate cell invasion. Experimental Design: Two independent patient cohorts of high-grade, invasive ductal breast cancer were interrogated for their expression of key EMT proteins using quantitative immunofluorescence. This analysis was extended to paired lymph node metastases for a subset of cases. EMT-related cell lines were selected based on gene and protein expression data. These lines were investigated using lightmicroscopy, immunohistochemistry and immunofluorescence in a three-dimensional assay that models invasion across the basement membrane. Results: Two transcriptionally-driven EMT programmes were identified. One comprises vimentin, Snail and Slug and is uncoupled from E-cadherin downregulation. A second is characterised by up-regulation of WT1, Snail and Slug and down-regulation of E-cadherin. Importantly, acquisition of this phenotype in lymph node metastases predicts poor outcome. Some aspects of these programmes were recapitulated in vitro. Conclusions: These results suggest that EMT does occur in human breast cancer but in a manner distinct to that seen in vitro. The examination of primary tumours with their paired lymph node metastases may significantly contribute to understanding EMT. Lastly, in vitro models can reflect aspects of tumour biology and may prove invaluable in identifying clinically relevant, targetable pathways.

2012/2013
High Mobility Group A (HMGA1a, HMGA1b and HMGA2) proteins are architectural nuclear factors, physiological expressed during embryonic development and re-expressed at high levels following neoplastic transformation, playing essential functions in both these processes thanks to their particular plasticity and consequently multifunctionality. HMGA are involved in a wide number of cellular processes, including Epithelial-Mesenchymal transition (EMT), a biologic developmental process characterized by the conversion of epithelial cells to motile mesenchymal ones, with increased capacity of migration and invasion. EMT plays a key role during the progression of different tumours, including breast cancer and also HMGA have been linked to these processes in the acquisition of tumourigenic features. Consequently taking advantage of different breast cancer cell lines to recreate an "EMT model" we have investigated the role of HMGA proteins in EMT and breast carcinoma. We have developed a cellular model, stable for the overexpression of HMGA1 using the human breast cancer cell line MCF7. We have explored different aspects of tumourigenesis, performing transwell migration and invasion assays, demonstrating that cells with high levels of HMGA1 migrate and invade at a higher and significant level in comparison to control cells. Moreover this data was also confirmed with the development of an inducible cell line for HMGA1 overexpression. Therefore we have examined the expression status of different genes, including several specific EMT markers at mRNA level with Real Time PCR, observing a pre-malignant change towards mesenchymal status. We have investigated the response after DNA damage induced by doxorubicin drug, by colony formation assay, demonstrating that HMGA1 overexpressing cells confer a survival advantage to the cells, being able to survive and form a significant higher number of colonies in respect to control cells. Therefore to study deeper the role of HMGA in EMT, we have developed other two cellular systems, a human cellular model of EMT in MDA-MB-468 human breast carcinoma cells treated with Epidermal Growth Factor (EGF) and the well known EMT model, elicited by Transforming Growth Factor-β (TGF-β) in murine mammary epithelial NMuMG cells, in which HMGA2 is functionally determinant. We have demonstrated by Real Time PCR of EMT markers, Western Blot analyses and immunofluorescence the effective reliability of these cellular models, confirmed also by a dramatic change in morphology of treated cells, towards a mesenchymal phenotype. Concluding we have interestingly observed that overexpression of HMGA1 could confer some tumourigenic features (i.e. migration, invasion) and survival advantage to the cells in the MCF7 model after a cellular DNA damage induction; therefore we have different suggestions that HMGA are involved in EMT in other different cellular models.
Le proteine HMGA (HMGA1a, HMGA1b e HMGA2), definite come fattori architetturali della cromatina, sono fisiologicamente espresse ad alti livelli nel corso dello sviluppo embrionale diminuendo gradualmente la loro espressione nel corso del differenziamento. Sono coinvolte, oltre all'aspetto fisiologico, anche in diverse condizioni patologiche, essendo ad esempio ri-espresse ad alti livelli nel corso della trasformazione neoplastica, esercitando funzioni essenziali grazie alla loro alta plasticità, alle peculiari caratteristiche biochimiche e conseguente multifunzionalità. Le proteine HMGA utilizzano diversi meccanismi per esercitare la loro funzione nell'acquisire capacità trasformanti, inclusa la transizione epitelio-mesenchimale. Questo processo biologico, primariamente identificato come fattore chiave dello sviluppo embrionale, è risultato di essere di fondamentale importanza anche nella trasformazione tumorale. Mediante questo meccanismo una cellula epiteliale, mediante molteplici cambiamenti genetici e biochimici acquisisce caratteristiche tipiche di uno "stato mesenchimale", caratterizzato ad esempio da un'aumentata capacità invasiva e migratoria. La transizione epitelio-mesenchimale esercita un ruolo chiave nel corso della progressione di diverse tipologie tumorali, incluso il cancro al seno, a cui in particolare anche le proteine HMGA sono state associate. L'obiettivo della Tesi è quindi quello di studiare il ruolo delle proteine HMGA nella transizione epitelio-mesenchimale e in particolare nel cancro al seno. A questo scopo abbiamo sviluppato diversi modelli cellulari di transizione epitelio-mesenchimale. Il primo modello ha previsto la creazione di un sistema stabile di over-espressione della proteina HMGA1 nella linea epiteliale di tumore al seno MCF7. Abbiamo analizzato diversi aspetti della tumorigenesi mediante saggi di migrazione ed invasione in transwell, dimostrando come alti livelli della proteina HMGA1 inducano un aumento di entrambi i processi rispetto ad una condizione di controllo. Inoltre i dati di migrazione sono stati confermati in un sistema inducibile per la over-espressione di HMGA1 nella stessa linea cellulare MCF7 e da saggi condotti in condizione di deplezione di HMGA1 attraverso strategie di silenziamento, dimostrando ulteriormente come la migrazione sia un fenomeno HMGA1 dipendente. Abbiamo inoltre esaminato lo stato di espressione di diversi geni, inclusi specifici marker di transizione epitelio-mesenchimale, mediante analisi di Real Time PCR, osservando un cambiamento verso una condizione di tipo pre-maligno e di parziale transizione ad uno stato mesenchimale. Inoltre è stata verificata la risposta al danno indotto da doxorubicina mediante saggio di colony formation, dimostrando come cellule over-esprimenti HMGA1 possiedano un vantaggio in termini di sopravvivenza e di numero di colonie formate, rispetto alle cellule di controllo. Per approfondire ulteriormente il ruolo esercitato dalle HMGA nella transizione epitelio-mesenchimale, sono stati sviluppati altri due modelli cellulari, uno nella linea epiteliale umana di cancro al seno MDA-MB-468 trattata con EGF (Epidermal Growth Factor), l'altro nella linea cellulare murina mammaria di tipo epiteliale NMuMG, trattata con TGF-β (Transforming Growth Factor-β), in cui l'azione di HMGA2 è stato dimostrato avere un ruolo determinante. Mediante analisi di Real Time PCR di marker di transizione epitelio-mesenchimale, di Western Blot e di immunofluorescenza abbiamo dimostrato l'effettiva solidità di questi modelli cellulari, confermato anche dal fatto che è possibile apprezzare un consistente cambio morfologico verso un fenotipo mesenchimale e una concomitante over-espressione delle proteine HMGA. Da questi modelli è stato quindi possibile evincere come le HMGA siano coinvolte nell'acquisizione di caratteristiche di tipo tumorale anche mediante processi di transizione epitelio-mesenchimale e come questi modelli siano utili al fine di semplificare network molecolari.
XXV Ciclo
1984

La transició epiteli-mesènquima (TEM) és un procés en què cèl lules epitelials, immòbils i amb polaritat apico-basal transiten cap un fenotip mesenquimal o fibroblàstic. L'expressió del factor de transcripció snail1 és suficient per induir TEM en cèl lules en cultiu i és necessari per la majoria de les TEM fisiològiques descrites. Snail1 és un membre de la família de proteïnes amb dits de Zinc que reprimeix gens epitelials (com l'E-cadherina) a través de la unió directa a seqüències especifiques dels promotors anomenades caixes E i posterior reclutament de corepressors. La TEM també es caracteritza per l'activació de gens mesenquimals, però el mecanisme pel qual snail1 indueix l'expressió d'aquests és poc conegut.

En aquest treball demostrem que snail1 actua a nivell transcripcional per incrementar els nivells dels marcadors mesenquimals FN1 (fibronectina) i LEF1 (de l'anglès, lymphoid enhancer-binding factor 1) a través d'un mecanisme nou per aquesta proteïna de dits de Zn que no requereix ni caixes E ni unió directa a l'ADN. A més a més, mostrem que, per a dur a terme l'activació, snail1 coopera amb dos factors de transcripció ja descrits en relació a la TEM: beta-catenina i NF-kappa-B. Els nostres resultats també proven que l'expressió forçada de la E-cadherina evita aquesta cooperació i conseqüent activació gènica. A banda d'aquest mecanisme, també hem descrit que el factor de transcripció TFCP2c, que no havia estat prèviament relacionat amb TEM, és necessari per l'activació del gen FN1 induïda per snail1.
Epithelial-mesenchymal transition (EMT) is a cellular process by which no motile epithelial, apico-basal-polarized cells transit towards a motile mesenchymal front-backpolarized phenotype. Expression of the transcription factor snail1 is sufficient to induce EMT in cultured cells and it is required for most of the physiological EMTs described. Snail1 is a member of the Zn finger protein family that represses epithelial genes (such as E-cadherin) by directly binding to specific promoter sequences called E-boxes and subsequent recruitment of corepressors. EMT is also accompanied by activation of mesenchymal genes, however, little is known of how snail1 induces their expression.

In this work we provide evidence that snail1 acts at the transcriptional level to increase the levels of the mesenchymal FN1 (fibronectin) and LEF1 (lymphoid enhancer-binding factor 1) genes through a novel mechanism for this Zn finger protein that does not require neither E-boxes nor direct binding to DNA. Furthermore, we describe a cooperative action in such mechanism between snail1 and two transcription factors previously related to EMT: beta-catenin and NF-kappaB. Our results also show that restoration of E-cadherin levels prevents such cooperation and subsequent activation. In addition, we also demonstrate that TFCP2c, which had not been previously linked to EMT, is also required for snail1-induced transcriptional activation of the FN1 gene.

The Yes-associated protein 1 (YAP1) is an oncogenic transcriptional co-activator, which is negatively regulated by the Hippo signalling pathway. If the Hippo pathway is deregulated, YAP1 can translocate to the nucleus where it interacts with various transcription factors to drive transcription. Suppressing YAP1 as a therapeutic strategy has attracted considerable interest, especially since YAP1 and oncogenic RAS have been shown to interact in different tumour models. I evaluated the role(s) of YAP1 in transforming non-tumourigenic epithelial cells along the epithelial-mesenchymal transition (EMT) spectrum. Using a tetracycline-inducible expression system, I found that induced YAP1 overexpression in non-tumourigenic mouse Eph4 cells resulted in the upregulation of the mesenchymal markers, demonstrating a partial EMT. As a comparison, H-RAS overexpression in Eph4 cells resulted in E-CADHERIN relocalization away from the cell-cell junctions, also demonstrating a partial EMT. Co- expression of H-RAS and YAP1 resulted in a transition further along the EMT spectrum. However, YAP1 overexpression alone did not enhance cell migration or proliferation, whereas single overexpression of H-RAS did. Therefore, mutations which lead to overexpression of oncogenic RAS can be considered ‘driver’ mutations as they confer a significant tumourigenic potential to cancer cells. In contrast, although the upregulation of mesenchymal markers by YAP1 may also confer a survival advantage, YAP1 overexpression is not sufficient to trigger E-CADHERIN relocalization. Thus, mutations leading to YAP1 overexpression are neither ‘driver’ or ‘passenger’ mutations. Instead mutations leading to YAP1 overexpression are likely to represent an intermediate between a ‘driver’ and ‘passenger’ mutation. Hence I am referring to them as a ‘co-pilot’ of tumourigenesis.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Carcinomas are solid tumors arising from epithelial tissue, and account for the majority of cancer deaths in the United States. In most occurrences of carcinoma, it is the metastases that kill, not the primary tumor. The Epithelial-Mesenchymal Transition (EMT) provides a model by which tightly associated epithelial cancer cells can disseminate to distant sites. Many factors are known to trigger the EMT, but the extent to which the observed phenotypes represent a common process is unknown. There is also little appreciation of the extent to which EMT-inducing factors interact with one another or act on common or redundant pathways. In this study, I sought a common gene expression signature of the EMT by comparing five mesenchymal cell lines independently derived from the same parental epithelial line using different EMT-inducing factors. The resultant EMT core signature strongly suggested a common pathway is involved. Bioinformatics analysis revealed the transcription factor ZEBI to be a possible mediator of this common pathway. ZEB1 was found to be both sufficient to induce EMT and necessary for maintaining the mesenchymal phenotype in the same cells. ZEBI and miR-200 were known to reciprocally regulate each other, but their relative importance to the EMT phenotype had never been directly tested. I found that ZEB1 induced EMT regardless of miR-200c levels, thereby excluding the model in which miR-200c downregulation is a necessary step for the EMT. I also show evidence that EMT induced by the transcription factor Snail works at least in part through ZEB1.
by Kong Jie Kah.
Ph.D.

Epithelial-mesenchymal transition is reportedly important in loss of epithelial integrity and cell migration in inflammatory/infectious diseases and cancer. Since Gram negative anaerobic periodontal pathogens are well-recognized to induce intense inflammatory responses; the present study investigated their ability to induce EMT in vitro. A 2D chronic inflammatory model was developed using either the H400 oral keratinocyte cell-line or primary rat oral keratinocytes which were exposed to heat-killed Fusobacterium nucleatum, Porphyromonas gingivalis and Escherichia coli LPS for up to 8-days. EMT-associated changes were determined using semi-quantitative-RT-PCR, PCR-arrays, ELISA, scratch/transwell migration assays, immunocytochemistry/immunofluorescence, and transepithelial electrical resistance. Chronically stimulated cultures increased extracellular levels of the EMT regulatory cytokines, TGF-β1, TNF-α and EGF, whilst subsequent EMT-induction was indicated by up-regulation of mesenchymal markers, including vimentin and N-cadherin, and concomitant down-regulation of epithelial markers including E-cadherin and β-catenin. In addition, intracellular signaling activity of key EMT regulatory transcription factors, Snail-1 and NF-ĸB, increased following chronic bacterial exposure and was associated with enhanced cellular migratory activity and reduced epithelial barrier integrity. These results indicated for the first time that EMT may be involved in the compromised epithelial barrier function observed during periodontitis pathogenesis which may occur in response to prolonged local bacterial exposure.

Thesis (M.A.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Communication from stromal cells to tumors contributes to the progression of several carcinomas. Stromal fibroblasts, also referred to as cancer associated fibroblasts, in part through their production of secreted factors, promote epithelial-mesenchymal transition (EMT). EMT contributes to cancer progression by disseminating cells from the primary tumor and increasing these cells migratory capacity, an initial step in metastasis. Recently, several microarray studies have identified aortic carboxypeptidase-like protein (ACLP) as being significantly up-regulated in cancers, including prostate cancer and breast cancer, leading to the hypothesis that ACLP may regulate tumor progression and metastasis. To begin to test this hypothesis, this study first examined ACLP expression in a mouse mammary ductal carcinoma model and detected abundant ACLP expression in the cells surrounding the tumor. Cultured fibroblasts, derived from these tumors, readily expressed and secreted ACLP. To explore the functional contribution of ACLP to EMT in vitro we treated normal murine mammary gland epithelial cells (NMuMG) with recombinant ACLP (rACLP). In NMuMG cells, rACLP modulated the expression of epithelial-mesenchymal transition markers, Snail, fibronectin, occludin, and a-smooth muscle actin. Furthermore, rACLP treatment resulted in E-cadherin dissolution from the cell surface when compared with controls. These studies indicate that fibroblasts within a breast carcinoma express and may secrete ACLP, and in vitro data demonstrate that rACLP is capable of promoting EMT in normal epithelial cells. Therefore, ACLP may serve as an important mediator in the progression of cancer.

This thesis was a step forward in better understanding the involvement of cancer cell plasticity in the progression of prostate cancer to aggressive disease. These studies described for the first time the transcriptional landscape of epithelial-mesenchymal plasticity in prostate cancer which led to the discovery of a molecular signature capable of identifying high-risk patients. In addition, this project revealed new-found molecular targets that may regulate cancer cell plasticity, thus making them attractive therapeutic options for patients with prostate cancer.

Epithelial-mesenchymal transition (EMT) regulates the cellular processes of migration, growth, and proliferation - as well as the collective cellular process of tissue remodeling - in response to mechanical and chemical stimuli in the cellular microenvironment. Cells of the epithelium form cell-cell junctions with adjacent cells to function as a barrier between the body and its environment. By distributing localized stress throughout the tissue, this mechanical coupling between cells maintains tensional homeostasis in epithelial tissue structures and provides positional information for regulating cellular processes. Whereas in vitro and in vivo models fail to capture the complex interconnectedness of EMT-associated signaling networks, previous computational models have succinctly reproduced components of the EMT program. In this work, we have developed a computational framework to evaluate the mechanochemical signaling dynamics of EMT at the molecular, cellular, and tissue scale. First, we established a model of cell-matrix and cell-cell feedback for predicting mechanical force distributions within an epithelial monolayer. These findings suggest that tensional homeostasis is the result of cytoskeletal stress distribution across cell-cell junctions, which organizes otherwise migratory cells into a stable epithelial monolayer. However, differences in phenotype-specific cell characteristics led to discrepancies in the experimental and computational observations. To better understand the role of mechanical cell-cell feedback in regulating EMT-dependent cellular processes, we introduce an EMT gene regulatory network of key epithelial and mesenchymal markers, E-cadherin and N-cadherin, coupled to a mechanically-sensitive intracellular signaling cascade. Together these signaling networks integrate mechanical cell-cell feedback with EMT-associated gene regulation. Using this approach, we demonstrate that the phenotype-specific properties collectively account for discrepancies in the computational and experimental observations. Additionally, mechanical cell-cell feedback suppresses the EMT program, which is reflected in the gene expression of the heterogeneous cell population. Together, these findings advance our understanding of the complex interplay in cell-cell and cell-matrix feedback during EMT of both normal physiological processes as well as disease progression.

The role of epithelial mesenchymal transition (EMT) has been identified to influence many processes associated with cancer development; malignant transformation, invasion, resistance to therapeutics to name but a few. Despite this, little is known about the relationship between EMT and DNA damage response (DDR). This study aims to investigate how the EMT programs activated in different cancer cell backgrounds influence DDR. The understanding of this interrelationship will help in designing new therapies to combat forms of cancer in which EMT plays a role. Microarray data obtained in three different EMT cell models show a strong influence of EMT transcription factor (TF) expression on DDR genes. The role of DDR in the genome integrity allowed us to suggest that EMT is a determinant of genome instability. A novel link between ZEB2, cyclin D1 and DNA Ligase 1 has been identified, and we propose the expression of ZEB2 increases the concentration of unrepaired single strand breaks within cells. Further investigation reveals EMT-TFs reduce HR gene expression, suggesting that activation of EMT pathways may represent the mechanism underlying the formation of BRCAness phenotype in sporadic cancers. BRCAness predicts PARP inhibitor sensitivity; and this project identifies two cell models in which activation of EMT programs confers sensitivity to the PARP inhibitor olaparib. However, the role of EMT-TF in conferring sensitivity to PARP inhibition is not conclusive. Overall, this project has identified two potential sources of genomic instability imposed by the deficiencies in single strand and double strand break repair pathways, both enforced by EMT. This project concludes EMT does play role in DDR; and further investigation will be required to elucidate the exact mechanisms and therapeutic perspectives of these observations.

The epithelial-to-mesenchymal transition (EMT) is a critical mechanism during the process of normal embryonic development and wound healing that can be pathologically re-activated during cancer progression. We hypothesized that comparing the transcriptional programs of multiple EMT-driving transcription factors (EMT-TFs) would identify a common set of critical EMT effectors. After elucidating this common transcriptional program, the commonly upregulated RNA binding protein RBMS3 was chosen as a target for functional validation. RBMS3 was both necessary and sufficient for EMT and breast cancer progression, demonstrating the validity of focusing on common EMT-associated effectors. Finally, by evaluating the associations of multiple EMT-TFs with the tumor microenvironment in several solid tumor types, ZEB1 and a ZEB1-regulated transcription program was identified as uniquely associated with immune suppression and poor prognosis. In conclusion, this study significantly advances both the understanding of the mechanisms underlying EMT and the distinct associations of different EMT-TFs with tumor biology and the tumor microenvironment.

Prostate cancer (PCa) cells predominantly metastasize to bone and the complex crosstalk between PCa cells and osteoblasts (bone-forming cells) and osteoclasts (bone-destroying cells) leads to increased tumour growth and worsening of bone disease. Understanding the mechanisms of PCa bone metastasis can identify the aggressive fraction of PCa resulting in earlier intervention. The ability of PCa cells to express bone cell-specific features, termed osteomimicry, could potentially explain the osteotropic nature of PCa cells. The aim of this study was to determine the role of osteomimicry in the regulation of epithelial-mesenchymal transition (EMT) in bone metastatic prostate cancer cells. It was demonstrated that the osteoblast-specific marker alkaline phosphatase (bone/liver/kidney) (ALPL) was overexpressed in bone metastatic (ARCaPM), compared to non-metastatic (ARCaPE), human PCa cells. Knockdown of ALPL resulted in decreased cell viability, increased cell death and a change from mesenchymal to epithelial morphology in ARCaPM and PC3 cells, and increased CDH1 expression along with decreased migration in ARCaPM cells. Treatment with extracellular ATP also resulted in decreased viability, increased expression of epithelial markers (CDH1, KRT14) and decreased expression of mesenchymal markers (VIM, ZEB1), and reduced expression of ALPL in ARCaPM cells. Small RNA-sequencing identified microRNAs differentially expressed between ARCaPE and ARCaPM PCa cell lines: miR-373 expression was lower in ARCaPM compared to ARCaPE cells and its overexpression in ARCaPM cells resulted in a change to epithelial morphology, increased expression of the epithelial marker CDH1 and decreased expression of the mesenchymal markers VIM and ZEB1. Finally, the development of a high-throughput screening method to identify novel microRNA regulators of osteomimicry was described, which identified two microRNAs miR-199a-5p and miR-212 as positive regulators of ALP activity. Taken together, this thesis describes the identification of ALPL and ATP as novel regulators of epithelial-mesenchymal transition in PCa cells and high-throughput ALP-activity screening as a powerful tool to identify novel microRNA regulators of ALP expression.

Biochemistry
M.S.
Metastasis is a multi-step process that begins with cancer cells migrating and invading away from the primary tumor site and extravasating into distant organs to establish a secondary tumor. The loss of epithelial expression markers by neoplastic breast cancer cells in the primary tumor is believed to play a pivotal role during breast cancer metastasis. This phenomenon is the hallmark of the epithelial mesenchymal transition (EMT) process. Gene expression microarrays were performed to investigate key functional elements on an in vitro metastasis model derived from human breast epithelial cells (MCF-10F) treated with 17-beta estradiol. Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, and epithelial-mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, desmoplakin and various keratins were low, whereas SLUG, integrin beta 1 and fibronectin were high. SLUG, a zinc finger transcription factor acting as a transcriptional repressor, was defined as a promising target which led us establishing a SLUG-centered hypothetical pathway from the profile of dysregulated genes.
Temple University--Theses

Epithelial to Mesenchymal Transition (EMT) is a dynamic process by which a distinct change in the phenotype and function of epithelial cells render them as mesenchymal cells. Characteristics of mesenchymal cells include the ability to invade, increased migratory kinetics and heightened resistance to apoptosis. Therefore, there is a strong need to fully understand the mechanism for the induction of EMT in pathological conditions such as carcinoma progression. Recent advances highlight two pivotal contributors, soluble growth factor (gf) signals, and mechanical signals, in the process. However, to date, no clear mechanism exists linking the two in epithelial transdifferentiation. Transforming Growth Factor-β1 (TGF-β1), a gf known to induce EMT in breast cancer formation, induces EMT on rigid surfaces and apoptosis on compliant surfaces. It is our belief that a combination of mechanical signals, gf signals, and the type of extracellular matrix (ECM) proteins assembled by cells together drive the process of EMT. Here we investigated the role of the ECM protein fibronectin (FN) in EMT. Upon assembly into elastic, insoluble fibrils through cell-generated forces which become larger on stiffer surfaces, FN is able to serve as a gf delivery system. We examined the following hypothesis: Increased tissue stiffness drives FN assembly, which exposes cryptic binding sites for various gfs, such as TGF-β1, and creates a high concentration of these gfs at the cell surface, which in turn drives EMT. In this project we investigated three aims: (1) evaluate the effect of inhibiting FN fibrillogenesis and gf localization on TGF-β1-induced EMT, (2) assess the effect of TGF-β1 concentration on spatial patterning of ECM dynamics, cell phenotype and adherens junctional force, and (3) probe the role of the FN matrix in TGF-β1-induced spatial patterning of EMT. Results showed that both inhibition of FN fibril assembly and blocking the gf binding site on fibrils significantly attenuated the downstream effects of EMT. In microcontact patterns of epithelial colonies, increasing gf concentration led to spatial patterning of FN fibrils, cell phenotype and cell-cell junctional force. Elimination of FN fibrils effectively attenuated TGF-β1-induced spatial patterning. The knowledge acquired through these studies serves as an addition to an increasingly important body of work aimed at elucidating how physical changes within the microenvironment regulate physiology and pathology. By establishing a novel mechanism by which gf signaling induces EMT through interaction with the extracellular matrix, this research serves to combat the development and initiation of pathological phenomena, such as metastasis.

Understanding why some cancer cells do not respond to chemotherapy is critical to developing new ways to control cancer. This project defined the important role of tumour cell plasticity in the response of prostate cancer cells to chemotherapy drugs. Key proteins that control cell plasticity have emerged as promising theranostic targets that can be pursued to develop new approaches to improve outcomes for men with metastatic prostate cancer.

In the UK, 12,000 patients die from metastatic breast cancer annually. There is therefore an urgent need to identify the molecules that cause metastasis. Recent work has revealed a role for the RUNX family of transcription factors in the development of metastatic breast cancer. The RUNX proteins form active transcription factor complexes when bound by the heterodimeric partner CBFβ to regulate the expression of metastatic genes. Previous work from our laboratory has demonstrated that knockdown of CBFβ resulted in a decreased invasion capacity of the metastatic breast cancer cell line MDA-MB-231. Three-dimensional culture of MDA-MB-231 cells revealed that loss of CBFβ induces a mesenchymal to epithelial transition (MET). The aim of this project was to determine the role of the RUNX/CBFβ complex in maintaining the mesenchymal phenotype of metastatic breast cancer cells. The data presented show that the phenotype changes were accompanied by changes in EMT marker-gene expression, including Snai2, MMP9, and MMP13. Induction of CBFβ in the CBFβ-knockdown cells remarkably restored both the invasive capacity and the mesenchymal phenotype. Further analysis revealed that maintenance of the mesenchymal phenotype was dependent upon both CBFβ-partner proteins, RUNX1 and RUNX2. Taken together the data presented in this thesis demonstrate that RUNX/CBFβ complexes drive the epithelial to mesenchymal transition (EMT) in breast cancer cells. These findings are likely to be important in the development of potential therapies to inhibit the metastatic spread of breast cancer.

Prostate cancer is the most frequently diagnosed non-skin cancer and the third leading cause of cancer mortality among men in the US. Androgens are functionally required for the normal growth of the prostate gland and play a critical role in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development, and cancer cell metastasis. This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells and evaluate the effect of taxol chemotherapy on androgen signaling in prostate cancer cells in prostate cancer. The EMT pattern was evaluated on the basis of expression of the epithelial markers as well as cytoskeleton reorganization in respond to DHT (1nM) and/or TGFβ (5ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. The AR transcriptional activity was determined on the basis of prostate specific antigen (PSA) mRNA expression and the androgen-response element (ARE) luciferase reporter assay. The interaction of AR and tubulin was investigated using immunoprecipitation, immunofluorescence as well as introduction of a truncated AR in human prostate cancer cells. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and led to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. Our study also reveals that treatment of prostate cancer cells with Paclitaxel or Nocodaxol inhibits androgen-dependent, as well as androgen-independent AR nuclear translocation and activation potentially via targeting the interaction of AR and microtubule cytoskeletal structures. Our findings on multiple aspects of AR function in prostate cancer development and progression may enhance the understanding of AR targeting therapy being a double-sided sword in the context of tumor microenvironment. These studies provide new insights into the mechanism of action of chemotherapy agents and the development of therapeutic resistance within tubulin/microtubule repertoire in prostate cancer cells.

Squamous cell carcinoma of the head and neck (HNSCC) is an extremely common and devastating disease with a bleak prognosis. Despite intensive research, survival rates have not improved over the past 30 years principally due to untreatable recurrent/metastasising disease. Feline oral squamous cell carcinoma (FOSCC) is an equally common disease in cats with an even less favourable prognosis than humans. Human and feline squamous cell carcinomas share similar etiopathogenesis, molecular markers, tumour biology and treatment thus making FOSCC an excellent model for HNSCC. Epithelial to mesenchymal transition (EMT), under the direction microRNAs (miRNAs/mirs) could be a key driver in oncogenic transformation and chemoresistance. The aim of this study was to induce resistance to characterise the EMT/resistance phenotype and to investigate whether common miRNA-mediated pathways are present in HNSCC and FOSCC that drive this phenomenon. We used epidermal growth factor (EGFR)-inhibitor gefitinib to induce resistance in HNSCC and FOSCC and investigated the associated EMT-related molecular changes. In vitro and in vivo invasive and migratory properties of both species were explored to determine whether resistance and/or EMT status conferred a functional advantage. We determined the miRNA expression pattern during acquisition of resistance to gefitinib in both species by next generation sequencing and screened candidate miRNAs as potential therapeutics. We found that gefitinib-resistance produced a previously unrecognised biphasic response that consisted of two distinct phenotypes, a highly invasive mesenchymal phenotype during early resistance, and a more epithelial phenotype associated with established resistance. The biphasic nature of this transition may prove critical in establishing effective therapeutic targets and the timing of treatment to overcome resistance or in preventing local invasion or metastatic spread of squamous cell carcinoma. We found that the major anti-apoptotic PI3K/AKT pathway was activated in transitioning and resistant cells of both species as demonstrated upregulation of AKT, pAKT and c-FLIP together with inactivation of PTEN by phosphorylation. This indicates that avoidance of apoptosis may be a major pathway in resistance that could be targeted therapeutically. We showed that three miRNAs were differentially expressed in both gefitinib-resistant human and feline cell lines: miR-107 was downregulated, and miR-551b and miR-574 were upregulated. These microRNAs provide potential therapeutic targets in the fight against drug resistance in head and neck cancer although much further research needs to be conducted to elucidate the complex network of interactions that may be affected by targeting these powerful regulatory molecules.

Introduction: Epithelial-mesenchymal transition (EMT) is characterized by the loss of an epithelial phenotype and gain of a mesenchymal phenotype, i.e., migratory and metastatic properties. The EMT process is therefore characterized by a low expression of E-cadherin and high expression of mesenchymal markers (e.g., N-cadherin, snail and vimentin). It is stated that cells which have undergone EMT also gain stem cell features. Therefore, both EMT and stem cell phenotypes have been implicated in carcinogenesis and metastasis of tumour cells. Furthermore, EMT is regulated by small non-coding molecules (miRNAs) that either function as tumour suppressors or oncogenes (oncomirs). Tumour suppressor miRNAs reverse EMT while oncomirs activate it. Therefore, investigating the relationship between miRNAs and EMT is important in addressing metastasis of colorectal cancers (CRC). Aims and Objectives: The aim of the study was to determine the association between miRNA (miRNA-21 and miRNA-34a) expression levels and EMT in CRC. In addition, this investigation aimed to correlate miRNA and EMT data with clinicopathologic features of the study cohort. Methodology: A total of 100 CRC (including 8 known HNPCC cases) Formalin Fixed Paraffin Embedded (FFPE) tissue blocks and their corresponding H&E slides were collected from the archives of the Division of Anatomical Pathology at the University of Cape Town. Subsequently, the FFPE tissue blocks were sectioned at 3μm and IHC analysis of 4 EMT markers (E-cadherin, N-cadherin, snail-1 and vimentin) and 1 stem cell marker (CD44V6) was performed. The stains were then evaluated and scored by a pathologist. The IHC data were then correlated with clinicopathologic features. Furthermore, 59 cases (FFPE tissues and corresponding H&E slides) which included the 8 HNPCCs were randomly selected for miRNA analysis. The H&Es were examined by a pathologist to demarcate normal and tumour regions. RNA was then extracted from 59 tumours and 12 normal tissues using a High Pure FFPET Isolation Kit (Roche). Subsequently, cDNA was synthesized and qRT-PCR was performed to determine the expression levels of miRNA-21 and miRNA-34a. MiRNA-21 and miRNA-34a expression levels were ascertained using the relative quantification method. Moreover, the clinical significance of the two miRNAs was evaluated in relation to MSI status. Therefore, IHC analysis of MLH1, MSH2 and MSH6 mismatch repair proteins was performed on the Ventana platform. Statistical analysis was performed using Fisher's and Pearson's Chi Square tests in Stata 12 to correlate EMT and clinicopathologic data. Additionally, the Mann-Whitney non-parametric test in GraphPad prism 6 was used to determine miRNA-21 and miRNA-34a expression in relation to EMT and MSI data. Results: Our results showed low expression of E-cadherin in 77% of cases. In addition, there was decreased expression of N-cadherin and vimentin in 98% whilst snail-1 expression was decreased in 65% of the cases. Low expression of CD44v6 was also seen in 78% of the cases. There was no correlation between EMT/stem cell markers and clinicopathologic data. Furthermore, increased miRNA-21 expression was significantly associated with grade, lymph node metastasis and age of patients. There was a significant correlation between high miRNA- 21 expression and down-regulated snail-1 and N-cadherin expression. MiRNA-34a expression was not associated with any of the clinicopathologic features. In addition, high miRNA-34a expression was linked with low expression of snail-1 and CD44v6. Increased miRNA-21 expression was related with MSS tumours, whereas there was no relationship between miRNA- 34a and MSI status. Conclusion: Our investigation shows that there is an inverse association between miRNA (miRNA-21 and miRNA-34a) expression and two EMT (N-cadherin and snail-1) markers in our colorectal cancer cohort. Our data also show that both miRNA-21 and miRNA-34a cannot be used as biomarkers to determine progression of the cancer. Contrary to previous studies, our findings indicate that miRNA-21 does not activate EMT in this CRC cohort. However, similar to other studies our results confirm that miRNA-34a may be repressing snail-1 expression, thereby inhibiting EMT in the cancer.

Background: Non-response to neoadjuvant therapy is a significant challenge for clinicians managing solid cancers. This thesis aimed to determine whether Epithelial Mesenchymal Transition (EMT) was associated with non-response to neoadjuvant therapy in patients with locally advanced rectal cancer. Methods: Representative tissue specimens from the tumour invasive front of consecutive patients undergoing resection of rectal cancer from 2009-2011 were used. Patients with marked regression to neoadjuvant therapy were classified as responders with the remainder as non-responders. Markers of EMT included: reduced immunohistochemical expression of membranous E-cadherin, increased nuclear beta-catenin expression and tumour budding. In-situ-hybridisation was used to assess the expression of microRNA-200c (mir200c), an upstream master-regulator of EMT. Real-time polymerase chain reaction was used to quantitate expression of the gene for E-cadherin. Results: From 103 patients undergoing resection of rectal cancer, 69 received neoadjuvant chemoradiotherapy; 65% of these were non-responders. Reduced mir200c expression was significantly associated with higher T grade. Reduced membranous E-cadherin, increased nuclear beta-catenin and tumour budding individually predicted the presence of extra-mural vascular invasion. Reduced E-cadherin, nucleic beta-catenin, reduced mir200c and tumour budding were all significantly associated with non-response to neoadjuvant therapy (all p<0.001). Reduced E-cadherin and mir200c expression were both associated with reduced cancer specific survival (log-rank p-value 0.036 and 0.009 respectively). E-cadherin gene expression was not related to radiotherapy response or tumour budding. Conclusion: Targeted biomarkers of EMT were associated with non-response to neoadjuvant therapy and reduced survival in advanced rectal cancer. EMT may provide a practical clinical biomarker and novel therapeutic target, to improve the proportion of patients who respond to neoadjuvant therapy.

"Over the last decade, Epithelial-to-Mesenchymal Transition (EMT) has gained the attention of cancer researchers due to its potential to promote cancer migration and metastasis. However, the complexity of EMT intertwined regulation and the involvement of multiple signals in the tumour microenvironment have been limiting the understanding of how this process can be controlled. Cell-cell adhesion and focal adhesion dynamics are two critical properties that change during EMT, which provide a simple way to characterize distinct modes of cancer migration. Therefore, the main focus of this thesis is to provide a framework to predict critical microenvironment and de-regulations in cancer that drive interconversion between adhesion phenotypes, accounting for main microenvironment signals and signalling pathways in EMT. Here, we address this issue through a systems approach using the logical modelling framework to generate new testable predictions for the field.(...)"
Instituto Gulbenkian de Ciência (FCG-IGC)

上皮間充質轉化（EMT），作為重要的生理和病理事件，廣泛的參與胚胎發育、組織纖維化病變及腫瘤轉移的過程。這一顯著的細胞表型變化包括上皮細胞失去緊密連接和極性，上皮細胞呈現纖維細胞形態以及增強的細胞移動性。囊性纖維變性跨膜電導調節器（CFTR）是一種廣泛表達於上皮細胞的氯離子和碳酸根離子通道。研究證實，CFTR 的蛋白轉運與上皮連接的形成和功能有關，同時 CFTR 的表達受到 EMT 誘導因子 HIF-1 和 TGF-β 的反向調節。另外，CFTR 的表達和功能被證實參與 EMT 相關信號分子 Wnt 和 NF-κB活性的調節。基於上述發現，本研究旨在闡述 CFTR 與 EMT 的相關性。
CFTR 參與的腎上皮 EMT 以及後續的腎纖維化首先被關注。實驗表明，在腎上皮細胞（MDCK）中，小 RNA 介導的 CFTR 基因敲降或抑製劑引起的CFTR 通道功能缺陷均引起間充質細胞特徵的出現，包括纖維狀細胞形態、細胞連接分子 E-cadherin, ZO-1 和 Occludin 表達下調和間充質細胞標誌分子 Vimentin 和 N-cadherin 上調、上皮細胞跨膜電阻減低以及細胞遷徙能力的增強。有趣的是，在單側尿道結紮的腎纖維化模型中，CFTR 表達被顯著下調。同時，動物實驗證實一個最常見的 CFTR 分子突變（deltaF508 -/-）增加了單側尿道結紮導致的腎纖維化的程度。另外，在缺氧引起的 EMT 過程中CFTR 的表達顯著下調；同時，腎纖維化模型中，HIF-1 和 CFTR 的表達呈現負相關。結果提示，生理及病理的條件下，氧氣的調節可能作為 CFTR 下調及其後續事件的誘因。進一步實驗發現，CFTR 功能抑製或基因突變可以引起Wnt 的富集和 β-catenin 的細胞核轉移。基於以上的實驗結果，在腎纖維化的過程中，CFTR 參與了缺氧引起的 EMT 過程，並通過激活 Wnt/β-catenin 信號調節相關的下游因子。
第二部分集中探究了 CFTR 在癌細胞EMT 及腫瘤轉移中的作用及機制。實驗證實，在 TGF-β 誘導的腫瘤細胞 EMT 過程中，CFTR 表達被抑制。TGF-β 可能作為病理狀態下的調節因子，引起腫瘤細胞中 CFTR 表達下調及EMT。抑制 CFTR 通道功能或敲降其蛋白表達導致明顯的間充質細胞特徵，這一變化在不同來源的腫瘤細胞系中呈均一性。相對地，過表達 CFTR 引起細胞遷移和侵潤能力地顯著下降。在體實驗顯示，CFTR 表達與腫瘤的轉移能力呈現負相關。進一步機制研究證明，CFTR 通過調節多重的通路參與 EMT的過程。首先，uPA 的表達和活性受到 CFTR 的反向調節，並且這一調節作用是由激活的 NF-κB 介導的。其次，抑制 CFTR 通道功能引起 β-catenin 的細胞核轉移。
綜上所述，研究發現 CFTR 通過調節多重信號參與腎上皮及腫瘤細胞的 EMT。同時，研究顯示 CFTR 的表達和功能與腎纖維化及腫瘤轉移有關。此研究對相關疾病的診斷和預後具有潛在的提示作用。
Epithelial-Mesenchymal Transition (EMT) is an intricate process by which epithelial cells lose their epithelial characteristics and acquire a mesenchymal-like phenotype. It is essential for numerous physiological and pathological processes, such as embryonic development, tissue fibrosis and cancer metastasis. The dramatic phenotype changes of EMT include loss of tight junctions and polarity, acquisition of a fibroblastic morphology and increased motility. The cystic fibrosis transmembrane regulator (CFTR) is known as an anion channel and extensively expressed in a variety of epithelial cells. Interestingly, the apical membrane expression of CFTR is reported to be required for the normal organization and function of epithelial junctions. Moreover, EMT inducers, such as HIF-1 and TGF-β, are known to suppress the expression of CFTR in epithelial cells. In addition, CFTR has been reported to be associated with expression and/or activity of Wnt and NF- κB, key factors known to be involved in EMT. Thus, we hypothesized that CFTR might play an important role in EMT.
In the first part of the study, the involvement of CFTR in EMT of kidney epithelial cells and renal fibrosis was investigated. Our experiments revealed that suppression of CFTR by either inhibitor or knockdown induced EMT in Madin- Darby canine kidney epithelial cells (MDCK). This was accompanied by the appearance of fibroblastic morphology, with reduced expression of epithelial junction proteins E-cadherin, ZO-1 and occludin and accumulated expression of the mensenchymal markers vimentin and N-cadherin, as well as reduced transepithelial resistance (TER) and enhanced migratory ability. Interestingly, the expression of CFTR was found significantly down-regulated in unilateral urethral obstruction (UUO) kidney. In addition, CFTR mutant (deltaF508 -/-), the most common mutation found in CF patients, increased the risk of renal fibrosis in UUO model. Our results showed that the expression of CFTR down-regulated in hypoxia induced-EMT in MDCK, and the expression of hypoxia-sensitive transcription factor, HIF-1, is inversely correlated with CFTR in UUO kidney. Accumulation of Wnt and translocation of β-catenin were also observed in CFTR inhibitors-treated MDCK and deltaF508 -/- UUO mice. Taken together, these findings suggest that CFTR may be involved in mediating hypoxia-induced EMT by influencing the Wnt/β-catenin signaling contributing to renal fibrosis.
In the second part of the study, the role of CFTR in EMT during cancer metastasis and the underlying mechanisms were investigated. Recent studies have demonstrated that cancer cells may reinstitute properties of developmental EMT including enhanced migration and invasion. On the other hand, the reverse process, known as mesenchymal-to-epithelial transition (MET), has been implicated in forming a secondary metastatic tumor. Using various tissue-derived cancer cell lines including human colorectal cancer cell line LIM1863, human lung carcinoma cell line A549, and human breast cancer cell lines MCF7 and MDA-MB-231, we report that induction of EMT by TGF-β sharply reduces CFTR expression in various tissue derived cancer cell lines, while overexpression of CFTR can reverse the TGF-β- induced EMT phenyotype. Interfering with CFTR function either by its specific inhibitor or lentiviral miRNA-mediated knockdown mimicks TGF-β-induced EMT and enhances cell migration and invasion. Ectopic overexpression of CFTR in a highly metastatic cancer cell lines downregulates EMT markers and suppresses cell invasion and migration in vitro, as well as the ability of the cells to metastasize to the lung in vivo. The EMT-suppressing effect of CFTR is found to be associated with its ability to alter NF-κB targeting urokinase-type plasminogen activator (uPA) and the nuclear translocation of β-catenin. Taken together, the present study has demonstrated a previously undefined role of CFTR as an EMT suppressor in cancer.
In summary, our findings have demonstrated a regulatory role of CFTR in EMT in both normal kidney epithelial cell line and various cancer cell lines. We conclude that CFTR plays important roles in renal fibrosis and cancer progression/metastasis by modulating EMT process through multiple pathways. The insights afforded by these studies will provide critical new information about the function of CFTR as a suppressor of EMT, which may have potential application in diagnosis and prognosis of fibrosis and cancer.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Zhang, Jieting.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 136-150).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Epithelial-Mesenchymal Transition --- p.1
Chapter 1.1.1 --- Concept and features of EMT --- p.2
Chapter 1.1.2 --- Roles of EMT in development and diseases --- p.10
Chapter 1.1.3 --- The Regulators of EMT --- p.13
Chapter 1.2 --- Structure and function of CFTR --- p.18
Chapter 1.2.1 --- General structure and channel functions of CFTR --- p.18
Chapter 1.2.2 --- Gene mutations and CF --- p.18
Chapter 1.3 --- Potential role of CFTR in EMT --- p.20
Chapter 1.3.1 --- CFTR in formation of cell-cell junction and membrane polarity --- p.20
Chapter 1.3.2 --- CFTR and EMT inducers --- p.21
Chapter 1.3.3 --- CFTR and EMT related pathways and factors --- p.22
Chapter 1.4 --- Hypothesis and aim of the study --- p.22
Chapter Chapter 2 --- CFTR involves in hypoxia induced EMT in renal fibrosis --- p.24
Chapter 2.1 --- Abstract --- p.24
Chapter 2.2 --- Introduction --- p.25
Chapter 2.3 --- Results --- p.30
Chapter 2.3.1 --- Knockdown of CFTR induces EMT in MDCK --- p.30
Chapter 2.3.2 --- Inhibition of CFTR channel function induces EMT in MDCK --- p.30
Chapter 2.3.3 --- CFTR is downregulated during the process of renal fibrosis --- p.36
Chapter 2.3.4 --- CFTR defect increases the risk of renal fibrosis --- p.39
Chapter 2.3.5 --- Hypoxia/HIF-1α rather than TGF-β as the inducer of CFTR repression during EMT and renal fibrosis --- p.44
Chapter 2.3.6 --- CFTR as a negative regulator of Wnt/β-catenin signaling in renal epithelium --- p.51
Chapter 2.4 --- Discussion --- p.57
Chapter 2.5 --- Conclusion --- p.61
Chapter 2.6 --- Materials and Methods --- p.61
Chapter 2.6.1 --- Cell culture and treatments --- p.61
Chapter 2.6.2 --- Plasmids and transient transfection --- p.62
Chapter 2.6.3 --- Western blot analysis --- p.62
Chapter 2.6.4 --- Measurement of trans epithelial electric resistance --- p.64
Chapter 2.6.5 --- Wound-healing migration assay --- p.64
Chapter 2.6.6 --- Animals and Obstructive model --- p.64
Chapter 2.6.7 --- HE and Masson's trichrome stain --- p.65
Chapter 2.6.8 --- Immunofluorescent and immunohistochemistry staining --- p.65
Chapter 2.6.9 --- Statistical analysis --- p.66
Chapter Chapter 3 --- CFTR down-regulation mediates EMT during cancer metastasis --- p.67
Chapter 3.1 --- Abstract --- p.67
Chapter 3.2 --- Introduction --- p.67
Chapter 3.3 --- Results --- p.73
Chapter 3.3.1 --- Repression of CFTR during TGF-β induced EMT in cancer cells --- p.73
Chapter 3.3.2 --- Hypoxia does not have significant effect on CFTR expression --- p.78
Chapter 3.3.3 --- Repression of CFTR channel function induces EMT in cancer cells --- p.81
Chapter 3.3.4 --- Knockdown/overexpression of CFTR induces/inhibits EMT and malignant phenotypes --- p.84
Chapter 3.3.5 --- CFTR inhibits lung metastasis in vivo --- p.94
Chapter 3.3.6 --- Anti-metastatic effect of CFTR involves NF-κB targeting uPA --- p.104
Chapter 3.3.7 --- Correlation between CFTR and β-catenin --- p.112
Chapter 3.4 --- Discussion --- p.116
Chapter 3.5 --- Conclusion --- p.122
Chapter 3.6 --- Materials and methods --- p.122
Chapter 3.6.1 --- Cell culture and treatments --- p.122
Chapter 3.6.2 --- Lentiviral production and transduction --- p.123
Chapter 3.6.3 --- Plasmids and stable transfection --- p.124
Chapter 3.6.4 --- RT-PCR analysis --- p.124
Chapter 3.6.5 --- Western blot analysis --- p.126
Chapter 3.6.6 --- Immunofluorescence staining --- p.126
Chapter 3.6.7 --- Cell growth assay --- p.127
Chapter 3.6.8 --- Migration assay --- p.127
Chapter 3.6.9 --- Invasion assay --- p.128
Chapter 3.6.10 --- In vivo tumor growth assay --- p.128
Chapter 3.6.11 --- In vivo metastasis assay --- p.128
Chapter 3.6.12 --- Human EMT PCR array --- p.129
Chapter 3.6.13 --- uPA activity assay --- p.129
Chapter 3.6.14 --- Statistical analysis --- p.129
Chapter Chapter 4 --- General discussion --- p.130
Chapter 4.1 --- Normal function of CFTR in epithelial polarity and barrier function --- p.130
Chapter 4.2 --- Down-regulation of CFTR is associated with EMT-related diseases --- p.131
Chapter 4.3 --- CFTR functions as a central mediator of different EMT signals --- p.132
Chapter 4.4 --- Future directions --- p.134
Chapter 4.5 --- Conclusion --- p.135
References --- p.136
Declaration --- p.151

碩士
國立臺灣大學
藥理學研究所
97
AMPK is a serine/threonine protein kinase that serves as an energy sensor in all eukaryoic cells, regulating energy balance. Epithelial-Mesenchymal Transition (EMT) is a crucial process for cancer cells to acquire invasive and metastatic phenotype. Loss of E-cadherin is a hallmark of EMT. Thus, re-expression of E-cadherin could elicit inverse process Mesenchymal-Epithelial Transition (MET). In lung adenocarcinomas, TGF-β is a major inducer of EMT. In this study, we found that AMPK activators AICAR and 2-DG downregulated E-cadherin expression, while AMPK inhibitors Ara-A and Compound C reversed E-cadherin expression which was downregulated by TGF-β in lung adenocarcinomas. Importantly, RNA ineference-mediated knockdown of AMPK suppressed TGF-β induced EMT through upregulation of E-cadherin expression. Silencing of AMPK also upregulated E-cadherin mRNA expression, suggesting that AMPK could regulate E-cadherin gene transcription. Snail and Slug were E-cadherin transcriptional repressors. Silencing of AMPK downregulated the protein but not mRNA expression of Snail and Slug, indicating that AMPK might inhibit E-cadherin gene expression. Targeting AMPK may be a useful strategy to retard cancer cell invasion and metastasis.