O meduloblastoma (MB) é o tumor cerebral maligno mais comum na infância. A formação/progressão desta neoplasia foi associada a alterações moleculares, que inclui a desregulação da via de sinalização Wingless (Wnt), responsável pelo desenvolvimento embrionário. Além disso, as proteínas da família Aurora-quinases (A, B e C) têm sido amplamente estudadas, uma vez que a Aurora A e B foram encontrados hiperexpressas em diversas neoplasias, como o MB. Estudos recentes mostraram que existe uma associação entre a Via Wnt e as Aurora-quinases. No entanto, poucos trabalhos foram realizados para confirmar essa associação. Ademais, não existem trabalhos que relatem os efeitos do AMG900, um pan-inibidor de aurora-quinases, em MB, dando enfoque na regulação da via Wnt. Assim, o objetivo deste trabalho foi avaliar a modulação da via Wnt pelo inibidor AMG900 nas linhagens celulares de meduloblastoma pediátrico. Foram realizados os ensaios de PCR convencional, sequenciamento, qRT-PCR, transfecção transiente, ensaio clonogênico, Western Blot e ciclo celular. As linhagens celulares UW402, UW473 e ONS-76 não apresentaram mutações no éxon 3 do gene CTNNB1 (?-catenina) e no éxon 15 do gene APC. Não foi observada uma expressão significativa de CTNNB1, confirmando que as linhagens não possuíam a via Wnt ativa. Com isso foi necessário a transfecção transiente com a ?- catenina. Após este ensaio, houve um aumento da expressão de CTNNB1, Ciclina D1 e CMyc nas três linhagens, o que não ocorreu com as Auroras A e B. No ensaio clonogênico foi observado uma redução do número de colônias nas linhagens UW473 e ONS-76. Observou-se um aumento da expressão proteica da ?-catenina, da Aurora A e B na UW473, o que ocorreu somente com a ?-catenina na linhagem ONS-76. Após o tratamento com o AMG900 ocorreu uma diminuição da expressão proteica de ?-catenina, da Aurora A e B em ambas as linhagens. A transfecção não alterou o percentil celular em G2/M na UW402 e UW473. Já na ONS-76 houve um aumento significativo em G2/M, e o AMG900 potencializou esse bloqueio apenas nessa linhagem. Os resultados sugerem que pode haver alguma relação entre a inibição das proteínas Aurora-quinases e a expressão de proteínas da via Wnt. / Medulloblastoma (MB) is the most common malignant brain tumor in childhood. Tumor formation/progression has been associated to molecular alterations that include dysregulation of signaling pathway Wingless (Wnt), responsible for embryonic development. In addition, cell cycle proteins Aurora-kinase (A, B and C) have been widely studied since Aurora A and B were found overexpressed in many cancers such as MB. Recent studies show that there is an association between Wnt pathway and Aurora kinase proteins. However, few studies have been conducted to confirm this association. Moreover, there are no studies reporting the effects of AMG900 in MB, by focusing on the regulation of the Wnt pathway. The aim of this study is to evaluate Wnt pathway modulation by Aurora kinases inhibitor AMG900 in pediatric medulloblastoma cell lines. Conventional PCR, sequencing, qRT-PCR, transient transfection, clonogenic assay, Western Blot and cell cycle assays were performed. UW402, UW473 and ONS-76 cell lines did not present mutations in exon 3 of CTNNB1 gene and exon 15 of APC gene. There was no significant expression of CTNNB1 and their target genes in these cell lines, confirming that they did not have Wnt pathway activated. Considering this, transient transfection was necessary. After this trial, there was an increase in expression of CTNNB1 gene and its target genes Cyclin D1 and C-Myc in the three cell lines, which was not observed in Aurora kinases. Furthermore, in the clonogenic assay, a reduction in the number of colonies in UW473 and ONS-76 cell lines was observed. It was also observed an increase in ?-catenin protein, Aurora A and B in UW473 cell line, but not in ONS-76 cell line. However, after treatment there was a decrease in protein expression of ?-catenin, Aurora A and B in both cells. Transfection did not change the cellular percentile in G2 / M in UW402 and UW473. In ONS-76 there was a significant increase in G2 / M, and the treatment with AMG900 potentiated this block only in this cell line. Results suggest that there may be some relation between the inhibition of Aurora kinase protein and protein expression in Wnt pathway.

Des mutations du gène STK11 furent initialement décrites comme responsable du syndrome Peutz-Jeghers, dont la gravité est lliée à une incidence accrue d’apparition de tumeurs. Le produit de ce gène, la sérine/thréonine kinase LKB1, a une expression ou une activité catalytique réduite, voir perdue, consécutivement à des mutations somatiques dans plusieurs types de cancer mais principalement du poumon (30% des NSCLC). Cette kinase est considérée de ce fait comme un suppresseur de tumeur d’importance. Les mécanismes moléculaires responsables de sa propriété suppresseur de tumeur restent à identifier. En effet, alors que sa fonction dans le métabolisme cellulaire, au travers de l’activation de la kinase AMPK, fut longtemps privilégiée, elle est actuellement remise en cause au profit de sa fonction de régulatrice de la signalisation Wnt canonique. Mes travaux de thèse confortent cette éventualité dans le cas des tumeurs pulmonaires (NSCLC). En effet, parmi les deux complexes fonctionnels que forme LKB1 avec les pseudokinases STRADα ou β, mes résultats démontrent que seul celui impliquant STRADβ intervient dans la régulation de la voie Wnt. Pour cela, le complexe LKB1/STRADβ se localise au niveau du cil primaire et participe à l’activation de la kinase MARK3. Ces résultats, étayés par un modèle murin invalidé pour STRADβ ainsi que l’analyse, a posteriori, de bases de données transcriptomiques adossées aux données cliniques de patients atteints de NSCLC, suggèrent que l’activité suppresseur de tumeur de LKB1 est associée à sa localisation et à sa fonction au niveau du cil primaire en participant à l’activation de MARK3 et à la régulation de la signalisation Wnt canonique. / STK11 gene mutations were originally identified as responsible for the Peutz-Jeghers syndrome of which severity is mainly related to an increase incidence of tumor development. The product of this gene the serine/threonine kinase LKB1 gets its activity or its expression reduced, sometimes even lost, following somatic mutations in several types of cancer such as pancreas, liver but mainly from lung. Indeed, almost 30% of non-small cell lung carcinoma (NSCLC) does not express anymore or only an inactive form, has led to consider this kinase as tumor suppressor of importance. While there is no doubt of the involvement of its catalytic activity molecular mechanisms responsible for its tumor suppressor properties remain to be identified. Indeed, whereas its function as regulator of cellular metabolism through AMPK has been favor for a while, it is currently re-assess to benefit to its regulator function on canonical Wnt signaling. My thesis work, reinforce this eventuality in NSCLC. Indeed, among the two functional complexes formed by LKB1 through its association with STRADα or β pseudokinases, my results show that only the complex related to STRADβ is involved in the canonical Wnt pathway regulation. For that, LKB1/STRADβ complex localizes at primary cilia and participates to MARK3 kinase activation. These results strengthened by a STRADβ knockout mouse model and an a posteriori transcriptomic analysis of lung adenocarcinoma patient datasets related to their clinical records, suggest that LKB1 tumour suppressor activity is associated with its localization and its function at primary cilia participating in the activation of MARK3 and thus regulation of canonical Wnt signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22133号 / 生博第420号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 渡邊 直樹, 教授 千坂 修, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Esophageal adenocarcinoma (EAC) is still associated with poor prognosis, despite modern multi-modal therapies. New molecular markers, which control cell cycle and promote lymph node metastases or tumor growth, may introduce novel target therapies. Dickkopf-1 (DKK1) is a secreted glycoprotein that blocks the oncogenic Wnt/β-catenin signaling and its aberrant expression has been observed in many malignancies, including EAC. In this study, we investigated the biological role of DKK1 in EAC. Analysis of DKK1 and active β-catenin expression in human esophageal tissues confirmed a simultaneous DKK1-overexpression together with aberrant activation of β-catenin signaling in EAC in comparison with Barrett’s and healthy mucosa. To elucidate the molecular role of DKK1, the OE33 adenocarcinoma cells, which were found to overexpress DKK1, were subjected to functional and molecular assays following siRNA-mediated DKK1-knockdown. At the functional level, OE33 cell viability, proliferation, migration and invasion were significantly attenuated by the absence of DKK1. At the molecular level, neither DKK1-knockdown nor application of exogenous recombinant DKK1 were found to alter the baseline β-catenin signaling in OE33 cells. However, DKK1-knockdown significantly abrogated downstream Akt-phosphorylation. On the other hand, the Wnt-agonist, Wnt3a, restored the Akt-phorphorylation in the absence of DKK1, without, however, being able to further stimulate β-catenin transcription. These findings suggest that the β-catenin transcriptional activity in EAC is independent of Wnt3a/DKK1 site-of-action and define an oncogenic function for DKK1 in this type of malignancy via distinct activation of Akt-mediated intracellular pathways and independently of Wnt-axis inhibition. Taken together, DKK1 may present a novel therapeutic target in EAC.

Die Wundheilung im Zusammenhang mit der Aktivierung des Wnt-Signalweg und Type 1 / Type 2 Immunity wurde im Maus Modell und in-vitro an verschiedenen Zeitpunkten untersucht. Es hat sich herausgestellt, dass der Wnt-Signalweg während der Spätphase der Wundheilung aktiviert wird, also unter Type 2 Immunity. Diese Arbeit deutet darauf hin, dass die Wundheilung über eine Modellierung des Wnt-Signalweg beschleunigt oder verbessert werden kann, allerdings nur an spezifischen Zeitpunkten.:LIST OF ABBREVIATIONS........................................................................................ 5 INDEX OF FIGURES..................................................................................................7 INDEX OF TABLES.....................................................................................................8 SUMMARY.................................................................................................................9 1 INTRODUCTION..............................................................................................11 1.1 Cutaneous wound healing...............................................................................11 1.2 Phases of wound healing................................................................................11 1.2.01 Inflammation............................................................................................11 1.2.02 Proliferation..............................................................................................13 1.2.03 Remodeling..............................................................................................16 1.3 Wnt-signaling...................................................................................................17 1.3.01 Wnt-ligands biogenesis and receptors....................................................17 1.3.02 Canonical and non canonical Wnt signaling...........................................20 1.3.03 Canonical Wnt/ß catenin signaling..........................................................20 1.4 Wound healing and canonical Wnt/ß catenin signaling...................................21 2 AIMS OF THE STUDY......................................................................................25 3 MATERIALS AND EXPERIMENTAL PROCEDURES........................................27 3.1 MATERIALS.....................................................................................................27 3.1.01 Instruments..............................................................................................27 3.1.02 Software..................................................................................................27 3.1.03 Websites..................................................................................................28 3.1.04 Consumables...........................................................................................28 3.1.05 Media and Buffers...................................................................................29 3.1.06 Reagents..................................................................................................29 3.1.07 Stimulation reagents................................................................................30 3.1.08 Anesthesia and analgesia used for the full-thickness skin wound model.......................................................................................................................30 3.1.09 Kits...........................................................................................................30 3.1.10 Quantitative reverse-transcription PCR primers......................................31 3.2 EXPERIMENTAL PROCEDURES.....................................................................32 3.2.01 Mice.........................................................................................................32 3.2.02 Cell Lines.................................................................................................32 3.2.03 Cell counting with trypan blue stain and hemocytometer method.........32 3.2.04 Preparation of murine H133WT immortalized primary keratinocytes cultures....................................................................................................................33 3.2.05 Preparation of RAW264.7 murine macrophages cultures.......................33 3.2.06 Preparation of primary murine fibroblasts cultures.................................33 3.2.07 Type 1 and type 2 immunity cell stimulation...........................................34 3.2.08 Cell RNA isolation....................................................................................34 3.2.09 Mice wounding........................................................................................35 3.2.10 Whole skin tissue isolation......................................................................36 3.2.11 Epidermal and dermal tissue isolation....................................................36 3.2.12 Tissue RNA isolation...............................................................................36 3.2.13 cDNA synthesis.......................................................................................36 3.2.14 Quantitative Real-Time PCR (qPCR).......................................................37 3.2.15 Statistical analysis...................................................................................37 4 EXPERIMENTAL RESULTS..............................................................................39 4.1 Canonical Wnt/ß-catenin signaling activity in different skin cell populations under type 1 and type 2 immunity cytokines stimulation in vitro............................39 4.2 Canonical Wnt/ß-catenin signaling activity under type 1 and type 2 immunity conditions during wound healing in vivo.................................................................42 4.3 Wnt-ligands expression in different skin cell populations under type 1 and type 2 immunity cytokines stimulation in vitro.........................................................44 4.4 Wnt ligands expression in epidermal and dermal fraction under type 1 and type 2 immunity conditions in vivo..........................................................................51 5 DISCUSSION...................................................................................................53 6 CONCLUSIONS...............................................................................................59 7 PERSPECTIVES...............................................................................................61 8 REFERENCES..................................................................................................63 9 APPENDIX.......................................................................................................71 9.1 Supplementary results.....................................................................................71 9.2 Erklärung über die eigenständige Abfassung der Arbeit.................................73 9.3 Curriculum Vitae..............................................................................................75 9.4 Acknowledgments...........................................................................................79

The TCF/LEF transcriptional factors regulate the target genes of the Wnt signalling pathway - one of the key signalling mechanisms involved in development of multicellular organisms. The TCF/LEF genes produce a number of various protein isoforms, which consequently leads to a great functional diversity of the TCF/LEF proteins. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes, whose isoforms have not been studied yet. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels during seven developmental stages of C. elegans. Further, we also determined the expression profile of two important partners of pop-1, the bar-1 and sys-1 genes, whose protein products function as transcriptional co-activators. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1

For more than 20 years, T-cell specific factor 4 (Tcf4) is the most intensively studied member of the conserved Tcf/Lymphoid enhancer-binding factor (Lef) family of transcription factors. Together with β-catenin coactivator, Tcf4 represents the prominent nuclear effector of canonical Wnt signaling in the intestinal epithelium. Regulation of Wnt-β-catenin signaling in intestinal stem cells is crucial for tissue homeostasis and tumor formation initiation. Up to date, several mouse models were generated to manipulate Tcf4 abundance or activity in vivo and dissect its function. Moreover, mutational screens and expression profiling of human colorectal tumors were carried out to disclose a contribution of TCF4 to tumor progression. However, subsequent studies brought conflicting results in relation to the potential of Tcf4 to activate or repress Wnt target genes and drive or inhibit cell proliferation. Here in this study, we analyze publicly available datasets for global expression of TCF4 and its paralogs in human tissues and colorectal cancer (CRC) samples. Notably, we present newly generated Tcf4flox5 mouse with a conditional Tcf4 allele that can be used to eliminate expression of Tcf4 from two alternative promoters of the gene. Using this mouse strain we documented that Tcf4 loss led to the demise of...

The Wnt signaling pathway represents the principal evolutionarily conserved signaling cascade found in all multicellular organisms. It plays a key role not only in many processes during embryogenesis, but also in maintaining tissue homeostasis and regeneration. By contrast, mutations in genes encoding components of the pathway often result in increased activation of Wnt signaling and underlie onset of many human diseases, particularly cancer. The canonical Wnt signaling pathway is essential for proliferation and maintenance of the pluripotent state of intestinal stem cells and thus for homeostatic renewal of the intestinal epithelium. However, aberrant (hyper)activation of the Wnt signaling pathway is the initial step in development of intestinal neoplasia. Understanding the causes and identifying the consequences of the Wnt signaling hyperactivation is crucial for deciphering mechanisms leading to malignant transformation. Although the canonical Wnt signaling pathway has been the subject of scientific studies for several decades, all regulatory mechanisms and consequences of its hyperactivation have not been completely elucidated yet. During my PhD studies, I focused on understanding function(s) of some components and target genes of this signaling cascade. In this theses, results of my first...

<p>In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration.</p><p>Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.