CLCA2 is a p53-inducible transmembrane protein that is frequently downregulated in breast cancer. CLCA2 is a 943 amino acid type I transmembrane protein that is cleaved near amino acid 700 to produce a diffusible 100kD product. The N-terminus contains a hydrolase-like domain with well-conserved HEXXH zinc binding amino acid motif that was proposed to cleave the precursor auto-proteolytically. We investigate the auto-proteolysis of CLCA2 precursor. Using membrane extracts or purified protein from CLCA2-transfected cells, we show here that CLCA2 cleavage is catalyzed by zinc and inhibited by metal chelator EDTA. Moreover, an E165Q mutation in the metal binding site abolished processing without affecting stability or trafficking. The mutant could be cleaved by co-transfected wild type CLCA2, showing that the mutation had not caused an un-cleavable conformation and suggesting that it occurs in trans. Wild type CLCA2 was able to cleave CLCA2 E165Q mutant in vitro only after denaturation and renaturation, suggesting that a conformational shift is required for cleavage. The efficiency of cleavage increased steeply with increasing concentration of precursor, consistent with trans proteolysis but not cis or cleavage by another agent. Accordingly, CLCA2 molecules bearing different epitope tags formed a stable complex that could be co-immunoprecipitated. Cleavage appears to be specific within isoforms; CLCA1 was unable to neither cleave CLCA2 nor form a stable complex with it. Furthermore, cleavage causes a conformational shift: an N-terminal antibody that immunoprecipitates the precursor fails to precipitate the N-terminal product unless it is first denatured with ionic detergent. We found that cleavage is enhanced by p53 induction due to DNA damage, implying that the cleavage has functional consequences for stress response. Moreover, we found that HEK and MCF10A cells expressing the E165Q mutant had a higher proliferation rate than cells expressing wild type CLCA2, suggesting that the metalloprotease activity contributes to the anti-proliferative effect of CLCA2. Physiologically, CLCA2 plays an essential role in epithelial differentiation. It is induced during epithelial differentiation in immortalized human mammary epithelial cells (HMEC), and its knockdown causes epithelial to mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with Epithelial V-like Antigen 1 (EVA1) and confirmed by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 family. EVA1 resembles tight junction proteins called Junctional Adhesion Molecules (JAMs) by structure but we found by confocal analysis that EVA1 is localized the lateral interface at cell-cell junctions. Analysis of transcriptional profiles revealed that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype, and upregulated during epithelial differentiation. Like CLCA2, knockdown of EVA1 resulted in rapid EMT in immortalized HMEC. The interacting domains were delimited by deletion analysis, revealing that both the proteins interact via their transmembrane segments (TMS). The interaction was specific, as other transmembrane proteins did not interact with CLCA2 or EVA1. We also found that CLCA2 binds to ZO-1 and beta-catenin at its c-terminus but EVA1 does not. Interestingly, we found that EVA1 does interact with ZO-1 in the presence of CLCA2, indicating that these three form a complex at the cell-cell junctions that allows stabilization of belt-like adherens junctions (AJ). On the other hand CLCA2 may also stabilize adherens junctions by sequestering beta-catenin at the cell-cell junctions. These results indicate that CLCA2 plays a key role in maintaining epithelial differentiation via multiple ways. Either by binding to beta-catenin or forming a complex with EVA1 and ZO-1, it plays a pivotal role in maintaining epithelial differentiation. This explains the downregulation of both CLCA2 and EVA1 during tumor progression.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18513号 / 医博第3933号 / 新制||医||1006(附属図書館) / 31399 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 羽賀 博典, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

VIP is neurotransmitter with pleiotropic functions in mammals. It is expressed by a large number of tissues, including the CNS, PNS, innate and adaptive immune systems. VIP has two endogenous G-protein coupled receptors, termed VPAC 1 and VPAC2. VIP signaling through VPAC1 receptor has been documented to transactivate EGFR in healthy and cancerous cells leading to the activation of multiple downstream signaling pathways. EGFR signaling is a potent inducer of the master regulator EMT, called Snail1, which is a zinc-finger, transcription factor that is associated with downregulating epithelial markers like E-cadherin, while upregulating mesenchymal markers necessary for invasion and metastasis. We hypothesize that VIP upregulates Snail1expression in cancer cells. Our results showed that VIP treatment of epithelial cells increased Snail1 expression transiently at 1h and 4h then returned to basal levels at 24h. This research has implications in development of targeted therapies for cancer.

Transcription growth factor beta (TGFβ) mediated epithelial to mesenchymal transition (EMT) of lens epithelial cells (LEC) is known to cause posterior capsular opacification (PCO). In this work, I have focused on the TGFβ-induced EMT pathway governed by the cellular actin cytoskeleton dynamics. This study is the first to report the involvement of transcription co-factor myocardin related transcription factor-A (MRTF-A) in TGFβ-induced EMT in the lens. Using rat lens epithelial explants, I have conclusively established that in LECs, TGFβ induces nuclear migration of MRTF-A leading to induction of αSMA expression. Furthermore, I have manipulated the intracellular translocation of MRTF-A indirectly using actin binding drugs and established that inhibiting nuclear migration of MRTF-A reduces αSMA production by the cells. In addition, direct manipulation of MRTF-A using adenoviral vectors carrying modified gene constructs show that presence of functional MRTF-A construct in the nucleus is necessary to trigger αSMA expression by causing EMT. In order to understand the involvement of matrix metalloproteinase -9 (MMP-9) in this specific pathway, explants were treated with an MMP2/9 inhibitor and rhMMP9. I have established that rhMMP-9 does not significantly affect the intracellular migration of MRTF-A. Nevertheless, gene expression studies showed that MMP-9 induces the expression of MRTF-A. Taken together, I believe MMP-9 functions through a feedback mechanism controlling MRTF-A expression in the cell. However, the presence of MMP-9 is necessary but not sufficient for induction in MRTF-A nuclear translocation. Overall, the work presented in this thesis demonstrates for the first time the presence of MRTF-A in LECs and successfully shows that the intracellular translocation of MRTF-A as an integral part of TGFβ induced EMT. Therefore, in the future, MRTF-A may be used as a successful target molecule to inhibit in order to prevent EMT leading to PCO. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23768号 / 医博第4814号 / 新制||医||1056(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 武藤 学, 教授 松田 道行, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Epithelial-to-mesenchymal transition (EMT) is critical for embryonic development and wound healing, and occurs in fibrotic disease and carcinoma. Here, we show that EMT also occurs within the bulge, the epithelial stem cell (eSC) niche of human scalp hair follicles, during the inflammatory permanent alopecia, lichen planopilaris. We show that a molecular EMT signature can be experimentally induced in healthy human eSCs in situ by antagonizing E-cadherin, combined with transforming growth factor-β1, epidermal growth factor, and IFN-γ administration, which to our knowledge has not been reported previously. Moreover, induction of EMT within primary human eSCs can be prevented and even partially reversed ex vivo by peroxisome proliferator−activated receptor-γ agonists, likely through suppression of the transforming growth factor-β signaling pathway. Furthermore, we show that peroxisome proliferator−activated receptor-γ agonists also attenuates the EMT signature even in lesional lichen planopilaris hair follicles ex vivo. We introduce lichen planopilaris as a model disease for pathological EMT in human adult eSCs, report a preclinical assay for therapeutically manipulating eSC EMT within a healthy human (mini-)organ, and show that peroxisome proliferator−activated receptor-γ agonists are promising agents for suppressing and partially reversing EMT in human hair follicles eSCs ex vivo, including in lichen planopilaris.

This thesis introduces a comprehensive methodology to improve electromagnetic transient (EMT) modelling of power cables systems. Several improved modelling and validation techniques are proposed at the parameter estimation, time domain simulation and validation stages of the EMT modelling of transmission lines. A novel approach is developed to model sector-shaped cables in electromagnetic transient type programs. First, the applicability of elemental sub-conductor technique is extended to accurately calculate the frequency dependent impedances of sector-shaped cables. The derived admittance and propagation characteristics of the sector-shaped cable are fitted with rational functions using the method of vector fitting in an EMT-type program. The time domain simulations are validated with the numerical inverse Laplace transform method. A novel frequency domain approach is presented to model cascaded transmission systems. The procedure is based on obtaining four composite propagation functions representing the cascaded system. The performance of the technique does not diminish with increased number of cascaded segments and it preserves the intrinsic details of each line segment. This method is capable of modelling cascaded overhead lines or cables with different characteristic admittances and line lengths. This method can be used to validate EMT models of cascaded transmission systems. An improved generalized transmission line model is introduced which is capable of accommodating time steps greater than the travel time of the line. The time step of the conventional EMT models of transmission lines is constrained by the smallest travel time of the line. When the high frequency transients at the line terminations are not of interest, inaccurate nominal π equivalents are used with large time steps to reduce the computational burden. The proposed model not only is more accurate than the π equivalents, but also degenerates to the conventional frequency dependent EMT model when used with time steps smaller than the travel time. Therefore, the proposed model is highly convenient as it can be used for all types of EMT simulations without resorting to nominal π equivalents when the large simulation time steps must be used to reduce computational burden. / February 2017

Les cellules souches cancéreuses mammaires sont considérées d'initier et d'être responsable du développement des tumeurs. Un certain nombre de marqueurs ont été proposées, mais la question sur la définition de la CSC dans les cancers du sein RE+ reste ouverte. Nous avons utilisé les lignées MCF7 et T47D comme modèles RE+ et les SUM159 comme RE- pour rechercher les fractions cellulaires enrichies des CSC. Nous avons utilisé un panel de marqueurs tels qu'ALDH, CD44/CD24, combiné avec la formation de mammosphères. Les xénogreffes dérivées de patients (PDXs) de cancer du sein ont également été analysées. ALDH+ et/ou CD44+/CD24- définie des fractions cellulaires enrichies en cellules initiatrices de mammosphères et la capacité de différenciation dans les SUM159 et T47D, mais pas dans les MCF7. Nous avons demandé si cela pourrait être lié à différents statuts de p53. Dans ce but, nous avons généré des cellules MCF7shP53 et a montré une augmentation nette de fraction CD44+/CD24- dans ces cellules et une formation accrue de mammosphères. Cependant, les fractions triées ALDH+ ou CD44+/CD24- ont augmenté la formation de mammosphères d'un facteur 2 maximal. Nous voulions enrichir les CSC (basées sur l'initiation de mammosphères), les cellules en monocouche et en mammosphères sont exposées sous faible tension d'oxygène (<2%) pendant 7 jours, elles sont ensuite caractérisées sur la base de leur expression d'un certain nombre de marqueurs de différenciation soit par FACS soit par immunofluorescence. Dans les mammosphères des lignées RE+, les cellules ont passé à un phénotype bipotent CK5+/CK8+ et augmenté la fraction CD24+/CD44-. La même tendance a été observée parallèlement dans les cellules incubées en hypoxie. En outre, les sphères ne portent pas d'augmentation de la tumorigénicité. En conclusion, les cellules formant les mammosphères correspondent à un sous-ensemble avec un phénotype spécifique qui ne portent pas d'augmentation de la tumorigénicité. / Breast cancer stem cells (BCSCs) are believed to initiate and sustain tumor development. A number of markers have been proposed but open questions remain on the definition of CSC in ER+ breast cancer. We used MCF7 and T47D BCCL as ER+ models and the SUM159 BCCL for ER- to search for BCSC enriched subpopulations. We used a panel of markers such as ALDH, CD44/CD24, combined with mammosphere formation. Breast patient derived xenografts (PDX) were also analyzed. ALDH+ and/or CD44+/CD24- define fractions of cells enriched in mammosphere initiation and differentiation capacity in SUM159 and T47D, but not in MCF7. We asked whether this could be linked to different p53 statuses. To this aim, we generated MCF7-shp53 cells and showed a net increase in CD44+/CD24- in these cells and increased mammosphere formation. However, sorted ALDH+ or CD44+/CD24- cells increased mammosphere formation at most by a factor 2. We wanted to further enrich for CSC (based on mammosphere initiation), cultured 2D and mammosphere cells to low oxygen (<2%) for 7 days and characterized the cells on the basis of their expression of a number of differentiation markers by either FACS or immunofluorescence. In mammosphere of ER+ BCCL cells switched to a bipotent CK5+/CK8+ phenotype and increased the CD24+/CD44- fraction. Parallel findings were made with cells incubated under low O2. Furthermore, spheres do not bear increased tumorigenicity. In conclusion mammosphere forming cells correspond to a specific phenotypic subset of breast cancer cells that do not bear increased tumorigenicity.

Dans de nombreux cancers, l’expression du gène Fhit (Fragile Histidine Triad) estfréquemment altérée. Fhit est décrit comme un important gène suppresseur de tumeur de parson rôle pro-apoptotique et anti-prolifératif. Nous avons mis en évidence que, in vivo et invitro, la diminution de l’expression de Fhit est associée au caractère infiltrant des cellulestumorales bronchiques, ce qui suggère que Fhit pourrait être impliqué dans le processusd’invasion tumorale. Nous avons en effet montré que la surexpression et l’inhibition de Fhitinduisent respectivement une diminution et une augmentation des capacités migratoires etinvasives des cellules bronchiques. Nous avons aussi mis en évidence que Fhit contrôlel’invasion des cellules tumorales bronchiques en régulant l’expression d’éléments clés de latransition épithélio-mésenchymateuse (TEM) tels que l’organisation des jonctions serrées etadhérentes, l’expression des métalloprotéinases matricielles et de la vimentine. De plus, Fhitrégule la TEM via une cascade de signalisation impliquant le récepteur au TGF-β, lesrécepteurs à tyrosine kinase (RTK), Src, Erk et Slug. Le double rôle de Fhit en tant quesuppresseur de tumeur et d’invasion renforce l’idée que Fhit pourrait représenter un nouveaubiomarqueur d’agressivité tumorale et pourrait constituer une nouvelle cible thérapeutiquedans le traitement des cancers broncho-pulmonaires. / In many types of cancers, Fhit (Fragile histidine triad) expression is frequentlydecreased or lost. Fhit is described as a tumor suppressor gene by its ability to induceapoptosis and to inhibit proliferation of tumor cells. We have demonstrated that a low Fhitexpression is associated with in vivo and in vitro invasiveness of lung tumor cells. Then, wehave shown that Fhit controls the invasive phenotype of lung tumor cells by regulating keyelements of epithelial-mesenchymal transition (EMT) such as cell-cell adhesion molecules,matrix metalloproteinase and vimentin expression. Our results provide also evidence that Fhitcontrols EMT by regulating several signaling pathways implying TGF-βR, RTK, Src, ERKand Slug. The dual function of Fhit as a tumor and invasion suppressor gene strengthens theidea that Fhit could represent a new biomarker of aggressiveness of lung cancer and couldconstitute a new therapeutic target to limit tumor progression.

Câncer se destaca no contexto de patologias por ser uma das doenças que mais acometem mortes por ano, sendo caracterizada como um conjunto de doenças multifatoriais que tem em comum o crescimento desordenado de células que invadem tecidos e órgãos, podendo espalhar-se para outras regiões do corpo, dando origem às metástases. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem carácter invasivo e migratório, além de se tornarem mais resistentes às drogas. Durante este processo, ocorrem inúmeras alterações celulares que modificam a estabilidade proteica e/ou promovem sua translocação subcelular, o transporte de proteínas para a membrana, alterações no citoesqueleto e incluindo o envio de proteínas para degradação pelo proteassoma. A desregulação de fatores de transcrição e modificação pós traducional de proteínas são fatores que podem levar à EMT. Após a eficiente indução da EMT in vitro utilizando o inibidor de histonas deacetilase (SAHA) em células de adenocarcinoma de mama MCF-7, foram realizadas análises proteômicas envolvimento os inibidores relacionados ao sistema ubiquitina proteassoma, MG132 e P5091. A modulação por inibição de USP7 resultou em variação da expressão de diversas proteínas biomarcadoras da EMT (SNAIL, ?-Catenina, CDK1) e proteínas envolvidas no ciclo celular (P53 e CDK1). O estudo proteômico permitiu a correlação do processo da EMT por SAHA com as vias de modificações pós traducionais relacionadas ao sistema ubiquitina proteassoma, e ainda propõe USP7 como alvo de estudos detalhados para EMT com potencial proposta terapêutica / Cancer stands out in the context of pathologies because it is one of the diseases that most affect deaths per year, being characterized as a set of multifactorial diseases that has in common the disordered growth of cells that invade tissues and organs, being able to spread to other regions of the body, giving rise to metastases. An important step in the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of the epithelial phenotype and acquisition of the mesenchymal phenotype by the tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. During this process, numerous cellular alterations occur that modify the protein stability and/or promote its subcellular translocation, the transport of proteins to the membrane, changes in the cytoskeleton and including the sending of proteins for degradation by the proteasome. Deregulation of transcription factors and posttranslational modification of proteins are factors that can lead to EMT. After an efficient induction of EMT using the histone deacetylase inhibitor (SAHA) in MCF-7 breast adenocarcinoma cells, proteomic analyzes were performed involving inhibitors related to the ubiquitin proteasome system, MG132 and P5091. Modulation by inhibition of USP7 resulted in varying expression of various EMT biomarker proteins (SNAIL, ?-Catenina, CDK1) and cell cycle (P53 e CDK1). The proteomic study allowed the correlation of the SAHA EMT process with the posttranslational modifications pathways related to the ubiquitin proteasome system and also proposes USP7 as the target of detailed studies for EMT with potential therapeutic proposal