The Wilms' tumour suppressor gene Wt1 is a key regulator of embryonic development and tissue homeostasis. In humans, mutation in the gene may lead to childhood kidney cancer, severe glomerular kidney diseases, gonadal dysgenesis and, in rare cases, heart diseases. The importance of WT1 in embryonic development is related to its crucial function in the regulation of two cellular plasticity processes: the epithelial to mesenchymal transition (EMT) and the reverse process, the mesenchymal to epithelial transition (MET). WT1 expression persists during the waves of EMT and MET that generate certain mesodermal tissues. In fact, WT1 is a major regulator of both transitions and it is essential for the survival of mesenchyme progenitors. Furthermore, it has been proposed that WT1 is required for the derivation of progenitors from different mesothelia, possibly through an EMT. Progenitors expressing WT1 are believed to differentiate into different cell types, giving rise to coronary vasculature, adipocytes and hepatic stellate cells. In my PhD I aimed to investigate the instructive role of different WT1 isoforms. To address this, the first goal was to generate a single plasmid that would accommodate all necessary components of an inducible system, in order to derive cellular models for the inducible expression of WT1 single isoforms. Second, I aimed to understand the processes that the single variants were sufficient to drive. Therefore, I started with the establishment of two cellular models for the inducible expression of the main four isoforms of WT1 (with or without the exon 5 and/or the KTS, here referred as +/+, +/-, -/+ and -/-). I cloned different plasmids carrying doxycycline inducible WT1 isoforms and derived single stable clones in two epithelial kidney cell lines that do not express WT1: the MDCK and the IMCD3 cells. I then analysed the expression profiles of the clones, using either microarray or RNA sequencing, and performed cellular assays to characterize the cells after WT1 induction. Overall, WT1 induction did not affect cell growth, cell cycle, cell migration or anchorage independent growth, suggesting that the expression of WT1 in these two cell lines does not change their oncogenic potential. The expression analysis of the MDCK cells suggested that the induction of WT1 isoforms activates an inflammatory response, leading to the overexpression of several cytokines. Moreover, the -/+ isoform speciffically caused the upregulation of fibrotic markers and the rearrangement of the actin cytoskeleton. Interestingly, the expression of the mesothelial marker UPK3B increased following the induction of the -/+ isoform. Because the expression of the -/+ variant led to the most signifficant isoform-specific changes in both cell lines, I focused on this isoform for the validation of the transcriptomic data of the IMCD3 cells. I confirmed that the induction of WT1 -/+ in the IMCD3 cells leads to the upregulation of fibrotic markers, increases cell adhesion and activates the AKT and MAPK pathways. Moreover, there was a significant overexpression of different mesothelial markers and, importantly, of key regulators and markers of developmental processes, such as adipogenesis, skeletal and cartilage development, as well as angiogenesis. I then dissected the timing of expression of some specific markers and regulators, analysing the levels of the genes at different time points after WT1 -/+ induction. The preliminary results intimate that WT1 -/+ might induce epithelial cells in the direction of cartilage-skeletal tissue and fat, possibly through a mesothelial intermediate. The data also suggest that the induction of this isoform initiates an EMT, possibly followed by an MET, as the levels of expression of the differentiation markers and regulators increase. To validate the proposed instructive role of WT1, it will be of crucial importance to determine both RNA and protein levels of markers and regulators at even later time points, both in IMCD3 cells and in a model of inducible embryonic stem cells, which is currently under development. In the future, it will be important to address the relevance of these findings in vivo and to dissect the molecular mechanisms.

Breast cancer is the most common cancer in women and unspayed female dogs. The Epithelial to Mesenchymal Transition (EMT) is a process involved in embryogenesis, carcinogenesis, and metastasis. The Transforming Growth Factor- Beta (TGF-β) pathway and its associated transcription factors are crucial for EMT induction, during which epithelial cells lose their defining characteristics and acquire mesenchymal properties. EMT has been implicated as a driver of metastasis as it allows cells to migrate and invade different organs. Recent evidence indicates that cancer stem cells are required to establish metastatic tumours at distant sites, and that EMT may promote development of cancer cells with stem-cell characteristics, thus, the EMT pathway may be an important molecular determinant of tumour metastasis. The main objective of this project was to characterise TGF-β-induced EMT in breast cancer models. EMT was induced by TGF-β in human, canine and feline breast cancer cell lines, and confirmed by morphological changes and molecular changes at the protein level by Western blot analysis. Changes at the mRNA level were confirmed in human and canine mammary carcinoma cell lines by qRT-PCR; migratory properties were assessed by invasion assays in vitro in feline and canine mammary carcinoma cells. Importantly, we observed that feline and canine mammary carcinoma cells stimulated by TGF-β acquired stem cell characteristics including sphere-forming ability, self-renewal, and resistance to apoptosis, and also enhanced migration potential. Canine cells showed resistance to chemotherapeutic drugs after TGF-β stimulation. These data suggests a link between EMT and cancer stem-cells. Moreover, global changes in microRNA expression were mapped during TGF-β-induced EMT of canine mammary carcinoma cells. This gave significant insight into the regulation of EMT in canine cancer cells and identified several potential targets, which require further investigation. During EMT cells acquire migratory properties and cancer stem-cell characteristics, suggesting that EMT and the stem-cell phenotype are closely related during cell migration and metastasis, therefore making the TGF-β pathway a potential target for the development of novel therapies against cancer and its progression.

Resumo: Transição epithelial-mesenquimal (EMT) é a conversão de células epiteliais polarizadas para células migratórias com fenótipo fibroblasto-símile. A EMT está envolvida na progressão e metástase em diversos cânceres nos seres humanos, porém permanece a ser mais bem explorada na literatura veterinária. O objetivo desta pesquisa foi avaliar a imunoexpressão de proteínas relacionadas à EMT nos carcinomas mamários de cadelas (CMCs). Seis proteínas foram avaliadas por meio de imunoistoquímica em 94 amostras de CMCs. Tecidos mamários não neoplásicos de 17 cadelas e amostras de 9 tumores mamários benignos de cadelas foram avaliados de modo a determinar o perfil de imunoexpressão de Snai-1. Características anatomopatológicas foram comparadas com a imunoexpressão de proteínas relacionadas à EMT nos CMCs. A perda de proteínas epiteliais e/ou a aquisição de proteínas mesenquimais foi observada principalmente em neoplasias com evidência de invasão estromal; entretanto, somente foi observada significância estatística quando comparado S100A4 e invasão vascular. Snai-1 foi observado em células luminais de neoplasias simples malignas e em células mioepiteliais de tumores benignos ou malignos de caráter complexo, sendo também significativamente relacionado à baixa de expressão de Caderina-E. Conclui-se que a perda de proteínas epiteliais e/ou a aquisição de proteínas mesenquimais está associada com EMT e pode possuir importante papel na avaliação de CMCs. O padrão único de imunoexpressão de Snai-1 pode ajudar a distinção entre um adenoma e um carcinoma não metastático e aparenta estar relacionado à conversão de células mioepiteliais a um fenótipo mesenquimal completo. A perda de Caderina-E e citoqueratina e a mudança no padrão de imunoexpressão de Snai-1, Caderina-N, S100A4 e MMP-2 indica a ocorrência de EMT em carcinomas mamários de cadelas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Epithelial-mesenchymal transition (EMT) is defined as switching of polarized epithelial cells to a migratory fibroblastoid phenotype. EMT is known to be involved in the progression and metastasis of various cancers in humans, but this specific process is still little explored in the veterinary literature. The aim of this research was to evaluate the expression of EMT-related proteins in canine mammary carcinomas (CMCs). The expression of six EMT-related proteins in CMC of 94 female dogs was evaluated by immunohistochemistry. Additionally, mammary tissues from 17 female dogs with no history of mammary tumor development and from 9 bitches with benign tumors were evaluated in order to determine Snai-1 immunoexpression patterns. Anatomopathological characteristics were compared with the expression of EMTrelated proteins in CMCs. Loss of epithelial protein and/or acquisition of the expression of mesenchymal proteins were observed, particularly in tumors with evidence of stromal invasion; however, significance was only observed between the S100A4 and vascular invasion. Snai-1 was only expressed in luminal cells of histologically malignant tumors and in myoepithelial cells of benign and malignant complex tumors and was significantly related to E-cadherin loss. In conclusion, loss of epithelial proteins and/or the acquisition of mesenchymal proteins are associated with EMT and may have an important role in the evaluation of CMC patients. The unique immunoexpression pattern of Snai-1 could help to distinguish between an adenoma and a non-metastatic carcinoma and seems to be related to conversion of myoepithelial cells to a complete mesenchymal-like phenotype. Loss of E-cadherin and cytokeratin and change of immunoexpression pattern of Snai-1, N-cadherin, S100A4 and MMP-2 indicate the occurrence of EMT in canine mammary carcinomas and should result in an en bloc resection or a close follow-up. / Orientador: Noeme Souza Rocha / Coorientador: Rafael Malagoli Rocha / Banca: Deilson Elgui de Oliveira / Banca: Geovanni Dantas Cassali / Mestre

acase@tulane.edu / Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) whereby cells adopt a motile and invasive phenotype through loss of epithelial markers, namely Cadherin 1/E-Cadherin (CDH1), and acquisition of mesenchymal markers, such as vimentin (VIM) and Cadherin 2/N-Cadherin (CDH2). While MAPK/ERK1/2 kinase inhibitors (MEKi) have shown promise as antitumor agents in the preclinical setting, application has had limited success clinically. Activation of compensatory signaling, potentially contributing to the emergence of drug resistance, has shifted the therapeutic strategy to combine MEK1/2 inhibitors with agents targeting oncoproteins (RAF) or parallel growth pathways (PI3K). Conventional MAPK family members have been well-characterized in modulation of cellular processes involved in tumor initiation and progression, yet the role of MEK5-ERK5 in cancer biology is not completely understood. Recent studies have highlighted the importance of the MEK5 pathway in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast. Furthermore, elevated levels of ERK5 expression and activity observed in breast carcinomas are linked to worse prognosis in TNBC patients. The purpose of this work is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide rationale for combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance. / 1 / Van Hoang

Colorectal cancer (CRC) is the fourth most common cause of cancer related deaths in the UK with the prognosis dependent on the degree of tumour invasion and presence of metastasis at diagnosis. An important step in the invasion and metastasis of solid tumours is the loss of cell-cell junctions and the acquirement of a more motile mesenchymal phenotype which is facilitated by the epithelial-mesenchymal transition (EMT). The presence of EMT is linked with a more aggressive, invasive tumour and subsequent poor prognosis. In addition to roles in motility and invasion, EMT can induce a cancer stem cell phenotype in a subset of tumour cells. Cancer stem cells (CSCs) are a subpopulation of cells capable of self-renewal and maintaining a cellular population whilst displaying increased therapeutic resistance. Induction of EMT and CSCs can be regulated by common signalling pathways with expression of EMT transcription factors inducing CSCs expression. Understanding the signalling pathways regulating EMT and CSC formation in cancer is important for preventing of metastasis and combating therapeutic resistance. Aspirin’s role in cancer prevention has been established for a number of years with aspirin treatment reducing the incidence of CRC. Recently, evidence has emerged suggesting aspirin treatment may have post-diagnosis benefits and increase survival rates of CRC patients. A potential mechanism for the post-diagnosis benefit of aspirin is the inhibition of EMT and CSC formation which both facilitate tumour progression and metastasis. Aspirin has been demonstrated to suppress the migratory and invasive capacity of lung cancer cell lines by inhibiting EMT. Whilst aspirin has been shown to inhibit platelet-induced EMT in CRC, the direct effects of aspirin on EMT in CRC cell lines has not been established. I hypothesis that aspirin inhibits cell migration, invasion and EMT in CRC which results in a reduction in the CSC population and contributes to the clinical benefit of post-diagnosis aspirin. Using CRC cell lines, I have demonstrated that aspirin treatment inhibits cell migration, invasion, motility and promotes an epithelial phenotype. These results have been confirmed in human organoids and mouse intestinal adenoma in vivo models. Aspirin also promotes a budding phenotype in Apc deficient organoids and reduces expression of stem cell markers in both mouse and human tissue. Aspirin inhibits the mTOR and Wnt signalling pathways in vivo which have the ability to regulate EMT and CSCs although signalling dependency has not been determined. Regardless, aspirin is decreasing the cancer stem cell population and promoting a non-invasive epithelial phenotype which may explain some of the previously described post-diagnosis benefits.

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T20:57:18Z : No. of bitstreams: 1 salgado_bs_me_botfm.pdf: 482776 bytes, checksum: 23c491910cd4c7da0d422455a7e9b2df (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Transição epithelial-mesenquimal (EMT) é a conversão de células epiteliais polarizadas para células migratórias com fenótipo fibroblasto-símile. A EMT está envolvida na progressão e metástase em diversos cânceres nos seres humanos, porém permanece a ser mais bem explorada na literatura veterinária. O objetivo desta pesquisa foi avaliar a imunoexpressão de proteínas relacionadas à EMT nos carcinomas mamários de cadelas (CMCs). Seis proteínas foram avaliadas por meio de imunoistoquímica em 94 amostras de CMCs. Tecidos mamários não neoplásicos de 17 cadelas e amostras de 9 tumores mamários benignos de cadelas foram avaliados de modo a determinar o perfil de imunoexpressão de Snai-1. Características anatomopatológicas foram comparadas com a imunoexpressão de proteínas relacionadas à EMT nos CMCs. A perda de proteínas epiteliais e/ou a aquisição de proteínas mesenquimais foi observada principalmente em neoplasias com evidência de invasão estromal; entretanto, somente foi observada significância estatística quando comparado S100A4 e invasão vascular. Snai-1 foi observado em células luminais de neoplasias simples malignas e em células mioepiteliais de tumores benignos ou malignos de caráter complexo, sendo também significativamente relacionado à baixa de expressão de Caderina-E. Conclui-se que a perda de proteínas epiteliais e/ou a aquisição de proteínas mesenquimais está associada com EMT e pode possuir importante papel na avaliação de CMCs. O padrão único de imunoexpressão de Snai-1 pode ajudar a distinção entre um adenoma e um carcinoma não metastático e aparenta estar relacionado à conversão de células mioepiteliais a um fenótipo mesenquimal completo. A perda de Caderina-E e citoqueratina e a mudança no padrão de imunoexpressão de Snai-1, Caderina-N, S100A4 e MMP-2 indica a ocorrência de EMT em carcinomas mamários de cadelas... / Epithelial-mesenchymal transition (EMT) is defined as switching of polarized epithelial cells to a migratory fibroblastoid phenotype. EMT is known to be involved in the progression and metastasis of various cancers in humans, but this specific process is still little explored in the veterinary literature. The aim of this research was to evaluate the expression of EMT-related proteins in canine mammary carcinomas (CMCs). The expression of six EMT-related proteins in CMC of 94 female dogs was evaluated by immunohistochemistry. Additionally, mammary tissues from 17 female dogs with no history of mammary tumor development and from 9 bitches with benign tumors were evaluated in order to determine Snai-1 immunoexpression patterns. Anatomopathological characteristics were compared with the expression of EMTrelated proteins in CMCs. Loss of epithelial protein and/or acquisition of the expression of mesenchymal proteins were observed, particularly in tumors with evidence of stromal invasion; however, significance was only observed between the S100A4 and vascular invasion. Snai-1 was only expressed in luminal cells of histologically malignant tumors and in myoepithelial cells of benign and malignant complex tumors and was significantly related to E-cadherin loss. In conclusion, loss of epithelial proteins and/or the acquisition of mesenchymal proteins are associated with EMT and may have an important role in the evaluation of CMC patients. The unique immunoexpression pattern of Snai-1 could help to distinguish between an adenoma and a non-metastatic carcinoma and seems to be related to conversion of myoepithelial cells to a complete mesenchymal-like phenotype. Loss of E-cadherin and cytokeratin and change of immunoexpression pattern of Snai-1, N-cadherin, S100A4 and MMP-2 indicate the occurrence of EMT in canine mammary carcinomas and should result in an en bloc resection or a close follow-up.

Breast cancer remains the most common malignant disease in women worldwide. Despite the advantages of early detection and improved treatments, studies into the mechanisms that initiate and drive breast cancer progression are still required. Recent studies have identified RUNX1, which is an essential transcription factor for hematopoiesis, is one of the most frequently mutated genes in breast cancer patients. However, the role of RUNX1 in the mammary gland is understudied. In this dissertation, we examined the role of RUNX1 in both normal mammary epithelial and breast cancer cells. Our in vitro studies demonstrated that RUNX1 inhibits epithelial to mesenchymal transition (EMT), migration, and invasion, reflecting its tumor suppressor activity, which was confirmed in vivo. Moreover, RUNX1 also contributes significantly to inhibition of the phenotypes of breast cancer stem cells (CSC), which is responsible for metastasis and tumor relapse. We showed that Runx1 overexpression reduces the tumorsphere formation and cancer stem cell population. Overall, our studies provide mechanistic evidence for RUNX1 repression of EMT in mammary cells, anti-tumor activity in vivo and regulation of CSC-like properties in breast cancer. Our results highlight crucial roles for RUNX1 in preventing epithelial to mesenchymal transition and tumor progression in breast cancer. This RUNX1 mediated mechanism points to novel intervention strategies for early stage breast cancer.

The inevitable progression of advanced prostate cancer to castration resistance, and ultimately to lethal metastatic disease, depends on primary or acquired resistance to conventional androgen-deprivation therapy (ADT) and accumulated resistance mechanisms to evade androgen receptor (AR) suppression. Whereas the canonical androgen/AR signaling axis maintains prostate cell growth, differentiation and survival, in prostate cancer cells, AR adaptations that arise in response to ADT are not singular, but diverse, and include gene amplification, mutation and even complete loss of receptor expression. Collectively, each of these AR adaptations contributes to a complex, heterogenous, ADT-resistant tumor that culminates in prostate tumor cells transitioning from epithelial to mesenchymal states (EMT) and the development of metastatic castration-resistant prostate cancer (mCRPC). Here, we examined prostate cancer cell lines that model common CRPC subtypes, each with different AR composition, and focused on novel regulators of tumor progression, the Bromodomain and ExtraTerminal (BET – BRD2, BRD3 and BRD4) family of proteins, to test the hypothesis that each BET family member regulates EMT and underlying characteristics such as cell motility and invasiveness. We systematically manipulated the BET proteins and found that BRD4 regulates cell migration and invasion across all models of CRPC, regardless of aggressiveness and AR status, whereas BRD2 and BRD3 only regulate cell migration and invasion in less aggressive models that retain AR expression or signaling. We determined that BRD4’s contribution to this process occurs through the transcriptional regulation of AHNAK, SNAI1 and SNAI2, which are EMT genes linked to promotion of metastasis in a diverse set of cancers. Furthermore, treatment of CRPC cell lines with low doses of MZ1, a small-molecule, BRD4-selective degrader, inhibits EMT and metastatic potential. Overall, these results reveal a novel, BRD4-regulated EMT gene signature that may be targetable to treat metastatic castration-resistant prostate cancer.

El mal pronòstic en una neoplàsia epitelial està associada a l'adquisició de característiques mòbils o invasives per part de les cèl·lules canceroses. Aquesta transformació morfològica es denomina transició epiteli-mesènquima (TEM). Snail és un factor de transcripció implicat en aquest procés, responsable de reprimir l'expressió de l'E-cadherina. Aquest treball demostra que Snail té la capacitat de reprimir l'expressió de MUC1 i VDR a través de la seva unió directa a caixes de reconeixement situades en els diferents promotors proximals. A més, la sobreexpressió d'Snail en diverses línies cel·lulars provoca un augment dels nivells d'ARNm de ZEB1 i un increment de l'activitat del seu promotor. L'activitat del promotor mínim d'Snail i els seus nivells d'ARNm depenen de la senyalització d'ERK. Finalment, hem demostrat que Snail i WT1, un regulador positiu de l'expressió de l'E-cadherina, competeixen per unir-se al promotor de l'E-cadherina i regular la seva transcripció. / The poor prognosis in epithelial neoplasia is associated with the acquisition of motile or invasive properties by the cancerous cells. This morphological transformation is often referred to as epithelial to mesenchymal transition (EMT). The Snail transcription factor is involved in this process by repressing the expression of E-cadherin. In this study we demonstrate the capacity of Snail to repress both MUC1 and VDR transcription by direct binding to specific sequences within their proximal promoter. Moreover, Snail overexpression in several cell lines induces ZEB1 mRNA and increases its promoter activity. The activity of the Snail minimal promoter is dependent on the ERK signaling pathway. Finally, we have demonstrated that Snail and WT1, a positive regulator of E-cadherin expression, compete for the binding to the E-cadherin promoter in order to regulate its transcription.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24879号 / 医博第5013号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 後藤 慎平, 教授 渡邊 直樹, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM