Specific SNPs in intronic regions of the human TCF7L2 gene are associated with an elevated risk of T2D development and progression. Several investigations have suggested a role of TCF7L2 in pancreatic β-cells. Whether this transcription factor is indeed expressed in the pancreatic islets of rodent species, however, has been a controversial issue. Here, we found that TCF7L2 mRNA level was significantly lower in the pancreas compared to the gut or Ins-1 cell line. In addition, TCF7L2 mRNA abundance in the pancreas was decreased by insulin. Finally, both TCF7 and TCF7L1 but not LEF-1 could be detected in the mouse pancreas. mRNA abundance for these two transcription factors was also decreased by insulin, and the level of TCF7, TCF7L1, and TCF7L2 mRNAs could be down-regulated by HFD. We speculate that reduced expression of these TCF genes during hyperinsulinemia may alter the Wnt signalling pathway and therefore impair the function of β-cells.

Bone is a highly specialised living tissue and has both mechanical and metabolical functions. Remodelling of the bone ensures a healthy bone mass and is regulated by a trio of secreted proteins, namely receptor-activator of NFKB (RANK), receptor-activator of NFKB ligand (RANKL) and osteoprotegerin (OPG). OPG, a major regulator of osteoclastogenesis, bone resorption and vascular calcification, is produced by various cell types including mesenchymally derived cells, particularly osteoblastic cells. Wnt signalling also plays a role in maintaining healthy bone mass. Dickkopf- 1 (DKK-1) is a soluble inhibitor of Wnt signalling and its excessive expression contributes to bone loss in rheumatoid arthritis and multiple myeloma. Recently, NDKK-1 has been demonstrated to be over-produced in osteoblasts of patients with Paget's disease of bone (PDB). The osteoblastic cell lines MG63 and Saos-2 were subjected to a series of different growth factors, hormones and cytokines to investigate the production of OPG, DKK-1 and the expression of various Wnt proteins. These results demonstrate that during standard culture conditions, both OPG and DKK-1 production in osteoblastic cells depend on a factor present in serum. Serum deprivation resulted in the up-regulation of Wnt4 and Wnt11, while down-regulating the expression of Wnt7b. Serum-induced OPG and DKK-1 production and Wnt expression was found to be regulated via a number of different signalling pathways. OPG production and expression was stimulated by platelet-derived growth factor-AB (PDGF-AB) not only in MG63 and Saos-2 osteosarcoma cells, but also a mouse pre-osteoblastic cell line (MC3T3-E1) and human bone marrow stromal cells (BMSC). PDGF-AB was shown to act through the PDGF receptor, PKC, PI3K, ERK and P38 and not via NFKB or JNK. PDGF isoforms AA, BB and AB demonstrated a similar stimulation of OPG production. The importance of PDGF in fracture healing suggests a role for OPG production in countering bone resorption during the early phase of this process. BIO, an inhibitor of canonical Wnt signalling resulted in the down-regulation of DKK-1 and the up-regulation of WntSa. Phorbol ester (PE), a known stimulator of PKC resulted in the up-regualtion of DKK-1, Wnt4, WntTa and Wnt16. The effects of PE were inhibited by bisindolymaleamide but not staurosporine. DKK-1 production, but not expression, was observed to be stimulated by calcium along with an up-regulation of WntTb and a down-regulation of WntWa and Wnt11. Incubation of pre-stimulated cells with Triton-X demonstrated the ability of calcium to increase DKK-1 secretion. DKK-1 was shown to be significantly elevated in the serum of PDB patients compared to healthy controls and did not correlate with ALP levels. Immunohistochemistry demonstrated that DKK-1 production is increased in both osteoblasts and fibrotic cells within the marrow cavity in PDB patients compared to fracture callus. B-catenin was found to be localised to intercellular membranes of plump osteoblasts, demonstrating its alternate role as a cell adhesion protein. DKK-1 therefore may be a useful biomarker of PDB and that Dkk-1 may play a central role in the aetiology of PDB. In summary, the results presented in this thesis have investigated the ways in which OPG and DKK-1 production in osteoblastic cells can be modulated with various effectors and the effect of Wnt signalling. These results may therefore be beneficial to increase the understanding of bone biology, improve fracture repair and generate further research into the role DKK-1 and the osteoblast in the aetiology of PDB to enable improved treatments to be developed.

The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown. Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In paper I, genetic alterations in Notch1 and the Notch1 regulating gene CDC4 were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating Notch1 mutations in 39% of the lymphomas, suggesting that Notch1 is an important target gene for mutations in chemically induced lymphomas. While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes per se are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In paper II, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including Notch2, were transcriptionally downregulated. We also confirmed binding of Lef1 to Notch2 as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the Notch2 promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target cyclin D1. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines. In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases. / Studies on molecular genetic alterations in colorectal cancer

Yes / The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.

Die Wnt/ β-Catenin-Signaltransduktion nimmt eine exponierte Stellung in der kolorektalen Karzinogenere ein. Niclosamid ist ein Derivat der Salicylsäure, das bei Bandwurm- infektionen eingesetzt wird. Es konnte gezeigt werden, dass Niclosamid den Wnt/ β-Catenin-Signalweg moduliert. Ziel der vorliegenden Arbeit war es, den therapeutischen Einsatz des Niclosamids beim kolorektalen Karzinom zu evaluieren. Die Zellproliferation von kolorektalen Karzinomzelllinien (humane SW480 und SW620 Zellen sowie CC531 Zellen einer Ratte) und von Rattenfibroblasten wurde nach 12 und 24 Stunden Inkubation mit Niclosamid durch lichtmikroskopische Zellzahlbestimmungen beurteilt. Die Apoptoseraten wurden mit einem Zelltod-Assay ermittelt. Eine Immunfluoreszenzfärbung gab Aufschluss über das Expressionsmuster von aktivem β-Catenin. Die Promotoraktivität des LEF/ TCF-Transkriptionsfaktors wurde nach Transfektion mit TOPflash mithilfe eines Luciferase Assays analysiert. Die Genexpression von Wnt-modulierenden Faktoren (Bcl-9 und Wif1), von Komponenten des ß-Catenin- Degradationskomplexes (Axin2 und GSK 3β), von kanonischen Zielgenen (Met, MMP7 und Cyclin D1) und von c-jun als Schlüsselprotein des nicht-kanonischen Wnt/ JNK-Signalweges wurde in der RT-PCR untersucht. Auf Proteinebene wurden zur Bestätigung zusätzlich Western Blots mit Antikörpern gegen aktives β-Catenin und c-jun durchgeführt. Die Zellproliferation kolorektaler Karzinomzelllinien wurde dosisabhängig inhibiert, und Niclosamid führte zu Apoptose. Nach Inkubation mit Niclosamid kam es nicht zur Umverteilung von aktivem β-Catenin von der nukleären in die zytosolische Fraktion. Die Wnt-Promotor-Aktivität von LEF/ TCF wurde nach 12 Stunden Inkubation mit 10 und 50 μM Niclosamid jedoch signifikant gesenkt. Kanonische Wnt-Zielgene (Met, MMP7 und Cyclin D1) sowie der Koaktivator Bcl-9 wurden auf Transkriptionsebene gehemmt, während das nicht-kanonische Schlüsselprotein c-jun aktiviert wurde. Fasst man zusammen, so führt die Inkubation mit Niclosamid zu inhibitorischen Effekten auf kolorektale Karzinomzelllinien und zu einer reduzierten kanonischen Wnt-Aktivität. Diese Effekte können durch eine gestörte Formation des Triple-Komplexes aus Bcl-9, β- Catenin und LEF/ TCF und einer Aktivierung von c-jun und damit des nicht-kanonischen Wnt/ JNK-Signalweges bedingt sein. In in vivo-Untersuchungen beabsichtigen wir, in einem Tiermodell die Daten zu verifizieren und so den Einsatz des Niclosamids als Option für Patienten mit metastasiertem kolorektalem Karzinom weiterführend zu beurteilen.

The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation.

The tumour-associated stroma, consisting of fibroblasts, inflammatory cells, vasculature and extracellular matrix proteins, plays a critical role in tumour growth, but how it regulates cancer cell migration and metastasis is poorly understood. The Wnt-planar cell polarity (PCP) pathway regulates convergent extension movements in vertebrate development. However, it is unclear whether this pathway also functions in cancer cell migration. In addition, the factors that mobilize long-range signalling of Wnt morphogens, which are tightly associated with the plasma membrane, have yet to be completely characterized. Here, I show that fibroblasts secrete membrane microvesicles of endocytic origin, termed exosomes, which promote tumour cell protrusive activity, motility and metastasis via the exosome component Cd81. In addition, I demonstrate that fibroblast exosomes activate autocrine Wnt-PCP signalling in breast cancer cells as detected by the association of Wnt with Fzd receptors and the asymmetric distribution of Fzd-Dvl and Vangl-Pk complexes in exosome-stimulated cancer cell protrusive structures. Moreover, I show that Pk expression in breast cancer cells is essential for fibroblast-stimulated cancer cell metastasis. Lastly, I reveal that trafficking in cancer cells promotes tethering of autocrine Wnt11 to fibroblast exosomes. These studies further our understanding of the role of the tumour-associated stroma in cancer metastasis and bring us closer to a more targeted approach for the treatment of cancer spread.

The tumour-associated stroma, consisting of fibroblasts, inflammatory cells, vasculature and extracellular matrix proteins, plays a critical role in tumour growth, but how it regulates cancer cell migration and metastasis is poorly understood. The Wnt-planar cell polarity (PCP) pathway regulates convergent extension movements in vertebrate development. However, it is unclear whether this pathway also functions in cancer cell migration. In addition, the factors that mobilize long-range signalling of Wnt morphogens, which are tightly associated with the plasma membrane, have yet to be completely characterized. Here, I show that fibroblasts secrete membrane microvesicles of endocytic origin, termed exosomes, which promote tumour cell protrusive activity, motility and metastasis via the exosome component Cd81. In addition, I demonstrate that fibroblast exosomes activate autocrine Wnt-PCP signalling in breast cancer cells as detected by the association of Wnt with Fzd receptors and the asymmetric distribution of Fzd-Dvl and Vangl-Pk complexes in exosome-stimulated cancer cell protrusive structures. Moreover, I show that Pk expression in breast cancer cells is essential for fibroblast-stimulated cancer cell metastasis. Lastly, I reveal that trafficking in cancer cells promotes tethering of autocrine Wnt11 to fibroblast exosomes. These studies further our understanding of the role of the tumour-associated stroma in cancer metastasis and bring us closer to a more targeted approach for the treatment of cancer spread.

In human pregnancy, the feto-placental vessels are crucial for efficient materno-fetal transfer; hence they play a pivotal role in the pathogenesis of fetal growth restriction (FGR). We, as well as other research groups, have observed abnormalities in the FGR feto-placental vasculature, which, though inconclusive, were suggestive of a state of panhypovascularity. The goal of the work presented in this thesis was to investigate this. We hypothesised that the placenta may be panhypovascular in FGR due to failed angiogenesis; and enhancing angiogenesis in the placenta may improve fetal growth. Custom-designed techniques including advanced imaging, computer-aided analyses and tube-forming experiments were employed to compare feto-placental vessels and endothelial cells in placentas from normal and FGR-complicated pregnancies while aiming to answer two main research questions: (i) is the FGR placenta panhypovascular? (ii) can angiogenesis be induced or enhanced to improve placental vascularity?Findings include: (i) shorter arterial [p = 0.03 and 0.009 when data adjusted for placental surface area (PA) and weight (PW) respectively] and longer venous path [p = 0.05 and 0.03, adjusted for PA and PW respectively] in FGR placentas though no difference in the total number of arterial or venous branches, diameter, and tortuosity of the vessels compared to normal; (ii) altered angiogenic behaviour/response of FGR placental endothelial cells following in vitro pharmacological manipulation of WNT signalling; (iii) human placental endothelial cells are capable of regaining their angiogenic potential following withdrawal of WNT inhibition. These findings discount the hypothesis of panhypovascularity in FGR placentas, but identify additional, previously unreported, feto-placental vascular abnormalities associated with FGR. Also, the findings provide evidence that impairment of WNT signalling may play a role in defective angiogenesis and consequent dysvascularity in the FGR placenta. The evidence suggests the WNT pathway should be explored as a potential new target for therapeutic interventions to correct placental dysvascularity in FGR.

Polydendrocytes, or NG2 glia, are fourth type of glial cells in mammal central nervous system. In the adult brain, NG2 glia represent important cell type with respect to their role in gliogenesis and nervous tissue regeneration following injury. Ligands from the Wingless/Int (Wnt) family play key role in proliferation and differentiation of NG2 glia and they can also influence regeneration of nervous tissue after ischemia. The aim of this thesis was to elucidate the role of NG2 glia in neurogenesis and gliogenesis following ischemic brain injury and investigate the impact of Wnt signalling on the reaction of NG2 glia to this type of injury. To fulfil these aims, transgenic mouse strains with tamoxifen-inducible recombination, that enabled simultaneous expression of red fluorescent dye and either activation or inhibition of the Wnt signalling pathway in NG2 glia, were employed. To induce ischemic injury, middle cerebral artery occlusion model was used. Changes in differentiation and electrophysiological properties of NG2 glia were analysed using patch-clamp technique. Activation of the Wnt signalling pathway under physiological conditions and 7 days after ischemic injury led to increased differentiation of NG2 glia toward astrocytes, while 3 days after ischemic injury activation of this signalling...