京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21029号 / 医科博第90号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 山下 潤, 教授 湊谷 謙司, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

One of the numerous complications of obesity is infertility. Leptin has been shown to reverse infertility; however, exact mechanism is poorly understood. Recent evidence indicates Ecadherin/ β-catenin complex, which is a structural constituent of adherens junction, is expressed in the rat ovary during folliculogenesis. We hypothesized that systemic leptin modulates the expression of E-cadherin and β-catenin in dietary-induced obese infertile rats to reverse infertility. Female Sprague-Dawley rats were fed either regular chow diet (RCD) (n=6) or high fat diet (HFD) (n=14). Oestrus cycles were monitored daily until their cycles became irregular. 100 ug/ml of leptin was given intraperitoneally to HFD-fed rats (n=5) with irregular cycles. The control rats HFD (n=9) and RCD received saline. Leptin treatment restored regular estrous cycle and increased the expression of E-cadherin and β-catenin in all the 5 rats (HFD+Leptin). This could represent the mechanism by which leptin reverses infertility in obese infertile rats.

During C. elegans embryogenesis, the DD, DA, and DB motor neurons arise from left and right lineages, move towards the midline and intercalate into a single tract to form the ventral nerve cord (VNC). Recently, the non-canonical Wnt-planar cell polarity was shown to regulate cell intercalation during VNC assembly. Disruption of this pathway causes DD neurons to shift anteriorly along the anterior-posterior (AP)-axis. Here, we investigated the role of the canonical Wnt-β-catenin pathway in positioning neurons in the VNC. Mutations in canonical Wnt pathway components, including bar-1/β-catenin and pop-1/TCF, cause the anterior displacement of DD2 towards DD1. In contrast, disruption of the β-catenin destruction complex gene pry-1/Axin results in the posterior displacement of DD1 towards DD2. In order to determine where and when defects occur, we used fluorescent time-lapse imaging to follow DD, DA and RIG neuroblasts during embryogenesis. In wild-type, we found that RIGL and DA2 intercalate between DD1 and DD2 via T1-type cell neighbor exchanges. Dorsal-ventral (DV) constriction of the DD1 and DD2 cell junction results in these cells meeting at a central vertex, which then resolves when the RIGL and DA2 cell junction expands along the AP axis. The resolution of the central vertex results in the spatial displacement of DD1 and DD2 along the AP axis. However, in Wnt-β-catenin mutants, central vertex resolution defects result in decreased spacing between DD1 and DD2 that persist into adulthood.

β-catenin is a multifunctional protein that plays crucial roles in embryonic development, physiological homeostasis, and a wide variety of human cancers. Previously, we showed that in vivo targeted ablation of β-catenin in melanoma-associated fibroblasts after melanoma formation significantly suppressed tumor growth. However, when the expression of β-catenin was ablated in melanoma-associated fibroblasts before tumor initiation, melanoma development was surprisingly accelerated. How stromal β-catenin deficiency leads to opposite biological effects in melanoma progression is not completely understood. Here, we report that β-catenin is indispensable for the activation of primary human stromal fibroblasts and the mediation of fibroblast-melanoma cell interactions. Using coimmunoprecipitation and proximity ligation assays, we identified Yes-associated protein (YAP) as an important β-catenin-interacting partner in stromal fibroblasts. YAP is highly expressed in the nuclei of cancer-associated fibroblasts (CAFs) in both human and murine melanomas. Mechanistic investigation revealed that YAP nuclear translocation is significantly modulated by Wnt/β-catenin activity in fibroblasts. Blocking Wnt/β-catenin signaling in stromal fibroblasts inhibited YAP nuclear translocation. In the absence of YAP, the ability of stromal fibroblasts to remodel the extracellular matrix (ECM) was inhibited, which is consistent with the phenotype observed in cells with β-catenin deficiency. Further studies showed that the expression of ECM proteins and enzymes required for remodeling the ECM was suppressed in stromal fibroblasts after YAP ablation. Collectively, our data provide a new paradigm in which the β-catenin-YAP signaling axis regulates the activation and tumor-promoting function of stromal fibroblasts.

Der (Pro)Renin Rezeptor (PRR) ist ein hoch konservierter Transmembranrezeptor, der ursprünglich beschrieben wurde Renin und Prorenin zu binden. Durch Bindung an Renin und Prorenin beeinflusst der PRR das Renin-Angiotensin-Systems und induziert eine MAP-Kinase-Signaltransduktion. Teile des PRR sind assoziiert mit der vakuolären H+-ATPase (vATPase), welche wichtig für die Azidifizierung zellulärer Organellen ist. Kürzlich wurde eine neue Funktion des PRR für den WNT/β-catenin Signalweg beschrieben. Hier dient der PRR als Verbindungsglied zwischen den WNT Rezeptoren und der vATPase. Die Mechanismen der Funktionen des PRR sind noch nicht verstanden, aber es wird angenommen, dass der PRR in die Regulation verschiedenster zellulärer Mechanismen involviert ist. Es gibt bis jetzt keine biochemische und strukturelle Charakterisierung des PRR. In der vorliegenden Arbeit wurden strukturelle Studien mit verschiedenen Konstrukten des extrazellulären Teils des PRR durchgeführt. Alle PRR Konstrukte (hsPRR (170-303), hsPRR (101-257) und hsPRR (166-257)) zeigten eine alpha-helikale Faltung und konnten nicht an Renin oder Prorenin binden. Der hsPRR (101-257) liegt in einem Konzentrations- und pH-abhängigen Monomer-/Oligomerequilibrium vor, während der hsPRR (166-257) als Monomer/Dimerequilibrium vorkommt. Diese Daten bilden die Grundlage für weitere strukturelle und funktionelle Untersuchungen. Konditionelle KO Mäuse sind eine exzellente Methode, um die physiologische Rolle des PRR in vivo zu untersuchen. Eines der wichtigsten Proteine des Wnt/β-catenin Signaltransduktionsweges, β-catenin, ist fundamental für die T-Zell Entwicklung. Aus diesem Grund wurde untersucht, ob die Deletion des PRR in T-Zellen ebenfalls zu einem Verlust von T-Zellen und zu einer Entwicklungsstörung führt. Die Ergebnisse zeigen, dass der PRR wichtig für eine vollständige T-Zellentwicklung ist und unterstützen die Hypothese, dass der PRR eine Rolle für Wnt/β-catenin Singaltransduktion in T-Zellen spielt. / The (pro)renin receptor (PRR) is an evolutionary conserved transmembrane receptor that was first discovered to bind renin and prorenin. Upon binding, PRR was shown to influence the activity of the renin-angiotensin-system (RAS) and to induce MAP kinase signalling. It was previously shown that a truncated, transmembrane part of PRR was associated to vacuolar H+-ATPase (vATPase), a proton pump which is important for acidification. Recently, a new function of PRR in the WNT/β-catenin signalling pathway was described. Here, the PRR was shown to be an adaptor between WNT receptors and the vATPase. The precise mechanisms by which PRR functions, are still elucidative but the PRR is supposed to regulate various cellular processes. Currently, no biochemical characterization or structural analysis is available for PRR. In order to gain understanding of the function of the PRR, structural studies were performed with several truncated proteins of the extracellular part of the PRR. All PRR proteins (hsPRR170-303, hsPRR 101-257 or hsPRR 166-257) showed an overall alpha helical folding and did not bind renin or prorenin. The oligomeric assembly of the proteins was investigated. The hsPRR (101-257) was shown to be in a concentration and pH dependent monomer/oligomer equilibrium, whereas hsPRR (166-257) is only present in a monomer/dimer equililibrium. These data are the basics for further structural and functional studies. Additionally, conditional KO animals are an excellent tool to investigate the physiological role of the PRR in vivo. As the major mediator of the Wnt/β-catenin signaling pathway, β-catenin, is crucial for T cell maturation, a conditionel deletion of PRR in T cells was analyzed. PRR deletion resulted in a loss of mature T cells. Moreover, a defect in T cell maturation in the thymus was determined. Our data showed that PRR is critical for proper T cell development and support the hypothesis that PRR contributes to Wnt/β-catenin signaling in T cells.

S100A4 spielt eine zentrale Rolle für die Metastasierung des Dickdarmkrebses. Die Hemmung der S100A4 Expression stellt damit einen vielversprechenden therapeutischen Ansatz dar. Die vorliegende Arbeit präsentiert Niklosamid und Calcimycin als neue Inhibitoren der S100A4 Transkription. In Kolonkarzinomzellen, die mit einem der beiden Inhibitoren behandelt wurden, wurde die S100A4 Expression konzentrations- und zeitabhängig unterdrückt. Des Weiteren war die Zellmigration und -invasion in Abhängigkeit von S100A4 in behandelten Zellen vermindert. Niklosamid und Calcimycin Behandlung verhinderten die Zellproliferation und die Koloniebildung von Kolonkarzinomzellen. Beide Inhibitoren hemmten den konstitutiv aktiven Wnt Pathway von Kolonkarzinomzellen. Calcimycin Behandlung verminderte die Expression von beta-catenin. Niklosamid hemmte die Bildung des beta-catenin/TCF Komplexes und unterband damit die Expression von Wnt Pathway Genen, wie z.B. S100A4. Im Rahmen dieser Arbeit wurde ein in vivo Tiermodell entwickelt, mit dem die S100A4-induzierte Metastasierung mit Hilfe von nicht-invasivem Biolumineszenz Imaging visualisiert werden konnte. In diesem Model konnte gezeigt werden, dass Niklosamid signifikant die S100A4 Expression im Tumor vermindert und damit die Metastasierung hemmt. Des Weiteren zeigt diese Arbeit, dass S100A4 die Expression des Wnt Pathway Antagonisten DKK-1 in Kolonkarzinomzellen hemmt. DKK-1 selbst konnte als endogener Inhibitor der S100A4 Expression identifiziert werden. Zusammenfassend beschreibt die vorliegende Arbeit einen neuen regulativen Mechanismus im Wnt Pathway, der die S100A4 Expression im Kolonkarzinom fördert. Diese Beobachtung verdeutlicht die Notwendigkeit für wirksame S100A4 Inhibitoren, wie Niklosamid und Calcimycin, die das Potenzial haben, in einer klinischen Anwendung die Metastasierung von Kolonkarzinompatienten mit erhöhter S100A4 Expression zu hemmen und damit deren Überlebenschance wesentlich zu verbessern. / S100A4 promotes metastasis in colon cancer patients thereby reducing their five-year survival chances to less than 10%. Consequently, inhibition of S100A4 expression is a promising strategy for anti-metastatic treatment of colon cancer patients. The present study characterizes the small molecules niclosamide and calcimycin as transcriptional inhibitors of S100A4 which reduced S100A4 expression concentration- and time-dependently. Niclosamide and calcimycin treatment restricted cell migration, invasion and wound healing capabilities in a S100A4-specific manner, and inhibited cell proliferation and colony formation of colon cancer cells. Both small molecule inhibitors interfere with the constitutively active Wnt pathway. Targeting β-catenin expression by calcimycin or interfering with the β-catenin/TCF transcription activating complex by niclosamide resulted in reduced Wnt target gene transcription, among them S100A4. The study further presents a human colon cancer xenograft mouse model for monitoring S100A4-induced metastasis formation via non-invasive bioluminescence imaging. Treatment of xenograft mice with niclosamide resulted in a significant reduction of the S100A4 mRNA level in the tumor accompanied by inhibition of metastasis formation. Moreover, this study presents evidence that S100A4 is an inhibitor of DKK-1 expression. In colon cancer cells DKK-1 and S100A4 expression was negatively correlated. Ectopic S100A4 overexpression inhibited DKK-1 expression. Targeting S100A4 via shRNA recovered the repressed DKK-1 expression and vice versa. In summary, the study describes a novel positive feedback loop in the Wnt pathway regulation formed by S100A4 repressing its antagonist DKK-1. This novel mechanism further strengthens the need for S100A4 inhibitors such as niclosamide or calcimycin. Consequently, such small molecules provide immense potential for the treatment of colon cancer patients who are at high risk for S100A4-induced colon cancer metastasis.

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Cirka 300 000 individer drabbas årligen i världen av oral cancer och mer än nittio procent av alla orala cancerformer utgörs av skivepitelcancer. Den femåriga prognosen är generellt 50 % och den 5-åriga relativa överlevnaden har under en tioårsperiod förblivit densamma. Detta motiverar utvecklingen av bättre prognostiska markörer och diagnostiska metoder för att tidigt identifiera de patienter som har risk att utveckla oral skivepitelcancer för att förbättra prognosen och minska lidandet genom tidig insatt behandling. β-catenin är en adhesionsmolekyl som är viktig för bibehållandet av cellulär integration och avvikelser i celladhesionsmolekyler tros spela en central roll när tumörceller invaderar närliggande vävnad det vill säga metastaserar till andra organ.Syftet med studien är att med hjälp av immunohistokemi undersöka om β-catenin kan fungera som en prognostisk markör för utvecklingen av oral skivepitelcancer. Detta görs genom att jämföra förekomsten av β-catenin med hjälp av monoklonala antikroppar i normalt skivepitel, dysplasi och cancer från 18 patienter som har diagnostiserats med oral skivepitelcancer. Infärgningen av Beta catenin jämfördes i normalt oralt skivepitel med cancer och dysplasi för alla biopsier för att undersöka om det förekommer någon skillnad av infärgningen. Förutom detta skedde även en jämförelse av normalt skivepitel med dysplasi och cancer inom varje enskild biopsi.Resultaten visade att det finns en skillnad i uttrycket av β-catenin i normalt skivepitel jämfört med dysplasi och cancer i denna patientgrupp. I denna studie visade mer än 70 % av biopsierna en stark eller måttlig och stark infärgning av β-catenin i normalt skivepitel, mer än 60 % av biopsierna visade en måttlig eller måttlig och svag infärgning av dysplasi och 58,8 % av alla biopsier visade svag infärgning eller ingen och svag infärgning av skivepitelcancer. Då studien visar att mängden av β-catenin är starkast i normalt oralt skivepitel, måttligt i dysplasi och svagast i cancer tyder detta på att β-catenin skulle kunna vara en viktig faktor i utvecklingen av skivepitelcancer i munhålan vilket stämmer väl överens med resultat från andra studier. / Approximately 300,000 individuals are affected every year in the world of oral cancer and more than ninety percent of all oral cancers consists of squamous cell carcinoma. The five-year prognosis is generally 50 % and the 5-year relative survival has over ten years remained the same. This motivates the development of better prognostic markers and diagnostic methods for the early identification of patients at risk of developing oral squamous cell carcinoma to improve prognosis and reduce the suffering of these patients with early treatment.β-catenin is an adhesion molecule that is important for the maintenance of cellular integration and abnormalities of cell adhesion molecules is thought to play a central role in tumorigenesis. The abnormalites is though to enhance tumour cells to break loose from neighbouring cells and invade nearby tissues and organs, however the exact mechanisms are unknown. The purpose of the study is that using immunohistochemistry to examine whether β-catenin may serve as a prognostic marker for the development of oral squamous cell carcinoma. This is done by examining the presence of β-catenin with monoclonal antibodies in 18 biopsies with normal squamous epithelia, dysplasia and cancer from 18 patients diagnosed with oral squamous cell carcinoma from the department of Oral Pathology at Malmö Högskola, Malmö. The staining of beta catenin was compared in normal oral squamous cancer and dysplasia for all biopsies to see whether there is any difference of dyeing. Besides this, there was also a comparison of normal squamous epithelium with dysplasia and cancer in each biopsy.The results showed that there is a difference in the expression of β-catenin in normal squamous epithelium, dysplasia and cancer in this population. In this study, more than 70 % of the biopsies expressed a strong or moderate and strong staining of β -catenin in normal oral squamous epithelium, more than 60 % of the biopsies showed a moderate or moderate and weak staining of dysplasia and 58.8 % of all biopsies showed weak or no staining and weak staining of squamous cell carcinoma.As the study shows that the amount of β -catenin is strongest in normal oral squamous epithelium, moderate in dysplasia and weakest in cancer, this suggests that β -catenin could be an important factor in the development of squamous cell carcinoma of the oral cavity which is in line with results from other studies.