The Wnt/β-catenin pathway is a fundamental regulator of embryonic development and adult tissue homeostasis. The key effector, β-catenin, is a multifunctional protein that occupies dual roles in signalling and intercellular adherens junctions. β-catenin primarily signals though the TCF/LEF transcription factors; however, many transcription factors, in addition to TCF/LEFs, interact with β-catenin, and the function of these interactions is poorly understood. To investigate novel β-catenin regulated signalling mechanisms with certainty, we developed TCF/LEF quadruple knockout (QKO) mESCs. In vitro differentiation of QKO cells reveals a neural differentiation bias, which is attenuated by overexpression of stabilized β-catenin. Our data indicate the presence of a TCF-independent β-catenin regulated neural differentiation blockade in mESCs. In addition to directly challenging the central dogma of canonical Wnt signalling, this finding has the potential to unveil new therapeutic targets for the treatment of many β-catenin-associated diseases, including forms of brain cancer that may arise from the oncogenic stimulation of neural stem cells. Furthermore, we describe an attempt to identify genome-wide TCF-independent β-catenin binding sites in QKO cells by ChIP-seq. Optimization trials provide proof of concept that the fold enrichment method of interpreting ChIP-qPCR results can be highly misleading when compared to the more comprehensive % input method of analysis. This conclusion has important implications for all fields of scientific research in which ChIP-seq methodology is employed. / Thesis / Master of Science (MSc)

La réponse antivirale innée constitue la première réaction d’une cellule à une infection virale. Il s’agit d’une réponse rapide, transitoire, non-spécifique, ubiquitaire qui a été conservée sous différentes formes au cours de l’évolution. La synthèse d’interféron β (IFNβ) joue un rôle essentiel dans l’établissement de la réponse antivirale innée chez les vertébrés. L’expression du gène Ifnb1 codant pour l’IFNβ est finement régulée au niveau transcriptionnel, ce qui permet de passer de la répression de son expression en absence d’infection à son activation après infection par un virus. Bien qu’une forte et rapide expression d’IFNβ soit bénéfique lors d’une infection virale, une expression anormale d’IFNβ peut entrainer des troubles physiologiques importants (réactions auto-immunes, dérégulations inflammatoires). En s’intéressant plus particulièrement à la séquence promotrice du gène Ifnb1, codant pour l’IFNβ, nous avons identifié deux sites de liaison potentiels pour les facteurs de la famille des TCFs (T-Cell Factor). En présence de β-caténine nucléaire, les complexes TCF/β-caténine se forment au niveau des sites de liaison aux TCFs et recrutent des complexes co-activateurs de la transcription. Dans une première partie de ce travail, nous avons étudié le rôle des complexes TCF/β-caténine dans la régulation de l’expression d’Ifnb1 aussi bien en absence que suite à une infection virale. Nous avons ensuite montré que ce mécanisme était fonctionnel car il permettait aux cellules traitées par un inhibiteur de GSK3 (qui induit une accumulation de β-caténine) de mieux se protéger contre l’effet cytopathique induit par le VSV. Lors de l’étude des fonctions biologiques ciblées par le Virus de la Fièvre de la Vallée du Rift (VFVR) auquel j’ai participé, il est apparu que cet arbovirus hautement pathogène ciblait via sa protéine non-structurale NSs, les promoteurs de gènes codant pour des nombreux acteurs de la voie Wnt/β-caténine et de l’adhésion cellulaire. Dans une deuxième partie de ce travail, j’ai analysé l’effet d’une infection par la souche pathogène (+NSs) ou non-pathogène (-NSs) du VFVR sur le taux de β-caténine et la présence de celle-ci au niveau des jonctions adhérentes. Les résultats obtenus montrent que l’infection par la souche pathogène de VFVR entraine une diminution du taux global de β-caténine ex et in vivo ainsi que la redistribution de celle-ci en dehors des jonctions adhérentes, couplée à une très forte désorganisation du cytosquelette d’actine de la cellule infectée. Cette perturbation du réseau d’actine est corrélée à la dérégulation de l’expression de certains gènes codant pour des protéines affectant la morphologie cellulaire. / No abstract available

Student No. 9404705H MDent Research Report School of Oral Sciences / Expression of the E-cadherin/β-catenin complex in oral squamous cell carcinoma and its correlation with histomorphology and metastasis. The immunohistochemical expression of E-cadherin and β-catenin was examined in 30 primary oral squamous cell carcinomas in patients with (n=19) and without (n=11) nodal metastasis, as confirmed on histopathological examination of the resected regional lymph nodes. The corresponding primary and nodal metastases tissue samples were available for 17 patients. The 30 primary carcinomas were histologically graded according to the invasive tumour front grading system and by conventional Broders’ criteria. None of the 30 primary carcinomas showed homogenous, membranous E-cadherin and β-catenin expression when compared to the normal oral squamous epithelium. Staining was heterogeneous in 73% (22/30) and in 77% (23/30) of the primary carcinomas stained for E-cadherin and β-catenin respectively. There was a highly significant reduction (P<0,001) of both E-cadherin and β- catenin expression with progression from the well-differentiated areas to the less differentiated tumour cells at the invasive tumour front. At the invasive tumour front, however, irrespective of the nodal status and invasive tumour front grading score, 28/30 (93%) tumours showed loss of E-cadherin expression. Loss of β-catenin expression was recorded in 22/30 (73%) cases. These findings indicate that E-cadherin and β-catenin play a key role in the loss of differentiation of tumour cells in oral squamous cell carcinoma and while they may be permissive for metastasis, in isolation, E-cadherin and β-catenin are probably not predictive of metastatic potential in oral squamous cell carcinoma.

ABSTRACT MUC1, a mucin-like transmembrane glycoprotein, is highly overexpressed and aberrantly localized in several invasive carcinomas. MUC1 is proposed to play numerous roles in the transformed behaviour of cells in which it is expressed. A number of these roles are facilitated by the interaction of MUC1 with β-catenin, a protein that is central to both cellular adhesion as well as Wnt-responsive gene transcription. The aim of this study was to investigate MUC1 expression, localization, and interaction with β-catenin, as a means of providing insight into the behaviour of human oesophageal squamous cell carcinoma. This cancer-type is exceptionally aggressive and is a major cause of cancer-related morbidity and mortality in South Africa. MUC1 is expressed and aberrantly localized in oesophageal squamous cell carcinoma cell lines, as demonstrated by RT-PCR, western blotting and indirect immunofluorescence. Moreover, evidence from coimmunoprecipitation assays shows that the MUC1 cytoplasmic tail and β-catenin form a complex both at the cell membrane and importantly, within the nucleus of these cell lines. This is the first demonstration of such a complex in the nucleus of a carcinoma derived from stratified, as opposed to simple, epithelia. Data presented here further indicates that activation of the epidermal growth factor receptor results in modulation of the association between MUC1 and β-catenin at the cell membrane. MUC1 membrane-localization, and interaction with β-catenin, may modulate cellular adhesion through steric interference of cell surface adhesion molecules as well as through sequestration of β-catenin away from adherens junctions. On the other hand, MUC1 association with β-catenin may enhance β- catenin signalling either through the stabilization of β-catenin, or as an essential functional component of the β-catenin/LEF/TCF transcription factor complex. Furthermore, results presented in this study identify oesophageal squamous cell carcinoma as a prime candidate for MUC1-specific immunotherapy. This finding is of substantial importance considering the ineffectual nature of existing therapies used in the treatment of oesophageal carcinoma.

1 / Rongrong Zhang

Die evolutionär konservierten morphogenen Signalwege Wnt/β-Catenin und Hedgehog (Hh) spielen vor allem in der Embryogenese, Zelldifferenzierung und Kanzerogenese eine große Rolle. Es ist bekannt, dass es zwischen Komponenten der beiden Signalkaskaden zu verschiedensten Wechselwirkungen und Hintergrundreaktionen in unterschiedlichsten Organismen und Geweben kommt. Ziel dieser Arbeit war die Aufklärung einzelner mole- kularer, transkriptioneller Prozesse hinter diesen Kreuzreaktionen in primären Hepatozyten und dem Hepatozellulären Karzinom. Dafür sind die beiden Signalwege durch verschiedenste Einflüsse, wie dem Einsatz von siRNAs, transgenen Mausmodellen und rekombinanten Proteinen gegen einzelne Faktoren der Hedgehog, aber auch der Wnt/β-Catenin Kaskade in ihrer Genexpression verändert und die Reaktionen der Signalwegskomponenten mittels der quantitativen Real-Time-PCR untersucht worden. Neben dem Organismus Maus haben einzelne vergleichende Experimente auch auf der humanen Ebene zum Erkenntnisgewinn beigetragen. Durch die Einflussnahme auf den Hedgehog Signalweg in murinen Hepatozyten wird deutlich, dass die Antworten auf die einzelnen Veränderungen in den Signalkaskaden sehr vielschichtig und umfangreich sind. Abhängig vom induzierten bzw. reprimierten Gen, aber auch vom gelenkten Signalweg variieren die Genexpressionen auf unterschiedlichste, und zum Teil gegenläufige Weise. Ferner wird deutlich, dass es zu Unterschieden in der Genantwort bezüglich der verschiedenen Organismen Maus und Mensch, aber auch zu Variationen der Interaktionen in den diversen Gewebe bzw. Zelltypen kommt.

La glande surrénale est un organe endocrinien d’une importance vitale de par son rôle dans le maintien de l’homéostasie corporelle. Pour assurer cette fonction, le cortex surrénalien est divisé en différentes zones qui produisent des hormones spécifiques. Les mécanismes de la mise en place de cette zonation au niveau embryonnaire ainsi que de son maintien tout au long de vie sont encore inconnus de nos jours. Les gènes Rspo1 et 3 sont exprimés de manière très spécifique au niveau de la capsule mais ils codent pour des protéines secrétées qui agissent sur les cellules adjacentes de la zone glomérulée afin d’induire l’activation de la voie canonique Wnt/β-caténine. La délétion du gène Rspo3 pendant le développement embryonnaire entraine un défaut d’activation des voies Shh et Wnt/β-caténine et donc en conséquence un défaut de la mise en place de la zonation. Sa fonction reste également essentielle au cours de la vie adulte puisqu’elle assure à la fois le maintien de l’homéostasie tissulaire et de la zone glomérulée. L’absence du gène Rspo1, n’affecte pas le développement ni la zonation ou l’homéostasie de la glande surrénale. Par contre, si on l’exprime de façon ectopique dans tous le cortex surrénalien, entrainant une activation anormale de la voie Wnt/β-caténine dans cette zone, on peut observer une hyperplasie des glandes surrénales. A partir de 1 an d’âge, cette hyperplasie surrénalienne entraine une formation de tumeurs. Ce travail démontre donc que la capsule par le biais du gène Rspondin 3 agit comme un centre de signalisation capable de contrôler à la fois l’homéostasie par le remplacement des cellules endommagés et le maintien de la zonation de la glande surrénale / The adrenal gland is an endocrine organ of vital importance because of its role in the maintenance of body homeostasis. To ensure this function, the adrenal cortex is divided into different areas that produce specific hormones. The mechanisms of the establishment of this zonation at the embryonic level as well as its maintenance throughout life are still unknown today. The Rspo1 and 3 genes are expressed very specifically at the capsule level but they encode secreted proteins that act on the adjacent cells of the zona glomerulosa in order to induce the activation of the Wnt / β-catenin canonical pathway. The deletion of the Rspo3 gene during embryonic development leads to a lack of activation of the Shh and Wnt / β-catenin pathways and hence a lack of zonation. Its function is also essential during adult life since it ensures both the maintenance of tissue homeostasis and the glomerular zone. The absence of the Rspo1 gene does not affect development, zonation or homeostasis of the adrenal gland. On the other hand, its ectopical expression in all the adrenal cortex leads to an abnormal activation of the Wnt / β-catenin pathway in this area and thus to an hyperplasia of the adrenal glands. From 1 year of age, this adrenal hyperplasia leads to the formation of tumors. This work demonstrates that the capsule through the Rspondin 3 gene acts as a signaling center capable of controlling both homeostasis by replacing damaged cells and maintaining the zonation of the adrenal gland

The liver is an essential organ for cholesterol homeostasis. If this process becomes dysregulated, cardiovascular disease (CVD) develops. Zinc-fingers and homeoboxes 2 (Zhx2) as an important hepatic gene regulator and contributes to CVD. BALB/cJ mice, with mutant Zhx2 allele, have fewer atherosclerotic plaques compared to other strains on a high fat diet. In my dissertation, I focused on the liver phenotype in BALB/cJ mice on a high-fat diet and found increased liver damage compared to wild-type Zhx2 mice. These data indicates that reduced Zhx2 in the liver leads to CVD resistance, but increases liver damage. Therefore, Zhx2 has an important role in lipid metabolism and liver function. Hepatic alpha-fetoprotein (AFP) is expressed abundantly in the fetal liver and repressed after birth regulated through three enhancers (E1, E2, and E3). E3 activity is restricted to a single layer of hepatocytes surrounding central veins (pericentral region) along with glutamine synthetase (GS). In my dissertation, I explore pericentral gene regulation in the adult liver. A GS enhancer (GSe) also exhibits pericentral activity which, along with E3, is regulated by the β-catenin signaling pathway. Orphan receptors, Rev-erbα, Rev-erbβ, and RORα, contribute to E3 activity elucidating a potential mechanism for zonation.

<p>Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.</p>

The AC133 epitope of the pentaspan transmembrane glycoprotein CD133 has been used as a cell-surface marker for normal and cancer stem cells from a broad range of tissue types. Despite the utility of CD133 as a marker, little is known regarding its regulation and biological function. To study these poorly understood aspects of CD133, I took two main experimental approaches: RNA interference (RNAi) screening and affinity purification coupled with mass spectrometry (AP-MS) to identify CD133 regulatory genes and CD133 protein-protein interactions (PPIs), respectively. Both of these experimental approaches relied on a human embryonic kidney (HEK) 293 cell line that exogenously expresses affinity tagged CD133 (HEK293/AC133). This cell line allowed me to perform a large-scale RNAi screen to interrogate 11,248 genes for their involvement in cell-surface AC133 recognition. This resulted in the identification of the N-glycosylation pathway as a direct contributor to CD133 plasma membrane localization and cell-surface AC133 detection. I used the same RNAi screening approach on the colon adenocarcinoma cell line Caco-2, which express CD133 from its native promoter, to identify factors that regulate endogenous CD133 transcription. I was able to demonstrate that AF4 promotes CD133 transcription in a number of cancer cell lines. Furthermore, I showed that CD133 expression in an acute lymphoblastic leukemia (ALL) cell line SEM, which is dependent on the mixed-lineage leukemia (MLL)-AF4 gene fusion, is critical for the viability of these cells. To gain further insight into the function of CD133, I performed AP-MS using HEK293/AC133 cells to identify CD133 PPIs. I identified histone deacetylase 6 (HDAC6) as a CD133 protein interaction partner. I found that HDAC6 negatively regulates CD133 trafficking into the endosomal-lysosomal degradation pathway. CD133 binds HDAC6 to prevent inhibition of HDAC6 deacetylase activity by phosphorylation. Protection of HDAC6 from phosphorylation promotes HDAC6 deacetylation of β-catenin, which results in β-catenin dependent signalling and the suppression of cancer cell differentiation. My thesis provide functional roles for CD133 as a pro-proliferative protein and as a key signalling protein in certain cancer cell lines.