Regulation of expression of the intestinal actin-binding protein, villin, a marker of intestinal epithelial differentiation, is poorly understood. Activation of the extracellular calcium-sensing receptor (CaSR) on sub-epithelial myofibroblasts stimulated the secretion of Wnt5a, while activation of the CaSR on intestinal epithelia increased expression of Ror2, a Wnt-family co-receptor. Immunocytochemistry has localized Ror2 expression in the epithelia lining the small intestine from the crypt base to the villus tip. The aim of this study was to determine whether Wnt5a binding Ror2 in intestinal epithelia stimulated transient increases in phospho-ERK1/2 (pERK1/2) which lead to increased expression of villin transcript and protein. To examine Wnt5a-Ror2 regulation of villin expression, we transgenically overexpressed wild-type, truncated, or mutant Ror2 constructs in HT-29 adenocarcinoma cells and nontransformed fetally-derived human intestinal epithelial cells (HIECs), added conditioned media containing Wnt5a and measured changes in ERK1/2 phosphorylation, villin amplicons and protein expression by RT-PCR and Western blot techniques. Wnt5a addition caused a transient increase in pERK1/2, which was maximal at 10 min but diminished by 30 min. Transient transfection with a siRNA duplex against Ror2 diminished Ror2 amplicons and protein and reduced the extent of pERK1/2 activation. Structure-function analysis revealed that deletion of the cysteine-rich, kringle, or tyrosine kinase domain or substitution mutations of tyrosine residues in the intracellular Ser/Thr-1 region of Ror2 prevented the Wnt5a-stimulation of pERK1/2. Deletion of the intracellular proline and serine/threonine rich regions of Ror2 had no effect on Wnt5a-stimulation of pERK1/2 in HT29 cells. Western blot analysis demonstrated that villin protein was increased by over-expression of wild-type Ror2 in HT-29 cells and HIECs in the presence of Wnt5a. The increase in villin expression was blocked by pharmacological inhibition of MEK1&2 and casein kinase 1, but not by PKC and p38 inhibitors. Neither Wnt3a nor EGF addition increased villin protein. This work suggested that stromal Wnt5a will stimulate pERK1/2 via the Ror2 tyrosine kinase domain to generate increased villin protein. These findings suggested that Ror2 homeostasis and Wnt5a interaction with Ror2 are important determinants of the regulation of villin expression in the intestine. / Thesis (Master, Physiology) -- Queen's University, 2009-07-14 23:34:39.397

La voie de signalisation WNT/PCP, couramment associée à la polarité planaire cellulaire, joue un rôle fondamental dans la morphogenèse chez les vertébrés. Parmi les différents composants protéiques de la voie WNT/PCP, on retrouve la protéine tyrosine kinase 7 (PTK7), dont les fonctions restent encore très peu décrites. Au cours de ma thèse, j’ai montré que PTK7 interagissait avec le récepteur tyrosine kinase ROR2. Ce complexe, après fixation du ligand WNT5A, induit la migration de fibroblastes embryonnaires murins via l’activation de JNK. Au cours du développement embryonnaire du xénope, Ptk7 interagit de manière fonctionnelle avec Ror2, et contrôle l’expression de la protocadhérine Papc ainsi que la morphogénèse. De plus, en utilisant une approche de Tissue MicroArray, réalisée sur des patients atteints de cancers colorectaux, j’ai pu montrer que PTK7 était retrouvé surexprimé chez 34% des patients, et que cette surexpression était un facteur de mauvais pronostic. Dans des lignées cellulaires issues de cancers colorectaux, la suppression de PTK7 par shRNA entraine une diminution de la migration des cellules tumorales, mais n’impacte pas leur prolifération et leur résistance aux drogues anticancéreuses. Dans un modèle de xénogreffe murin, la suppression de PTK7 induit une diminution du développement tumoral et l’expression de ce dernier, dans des cellules négative pour PTK7, entraine une augmentation de l’apparition de métastases chez les animaux injectés. Ce travail apporte de nouveaux éclaircissements sur le du récepteur PTK7 dans la voie de signalisation WNT/PCP, et le définit comme potentiel biomarqueur et cible thérapeutique dans le cancer colorectal. / The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. We show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells and physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a and that Wnt5a stimulates the release of the Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. Moreover, using a Tissue MicroArray produced from CRC patients we correlated PTK7 expression with pathological features and patient outcome. PTK7 was significantly up-regulated in CRC tissue, and its overexpression was found in 34% of patients. In CRC cell lines, shRNA PTK7 reduced migration, but did not affect cell proliferation and resistance to drugs. In a xenograft mouse model, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. This work reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements and define PTK7 expression as a potential prognostic biomarker and a novel therapeutic target in CRC.

Das Pankreaskarzinom geht nach wie vor mit einer sehr schlechten Prognose und hohen Letalität einher. Es hat mit 9 % die niedrigste 5-Jahresüberlebensrate unter den Krebserkrankungen und liegt damit an 4. Stelle der durch Krebserkrankungen verursachten Todesfälle. Aktuell ist der einzig kurative Therapieansatz die operative Resektion mit anschließender Chemotherapie. Die molekulare Pathogenese gestaltet sich sehr vielseitig und komplex. WNT5A gehört zur Familie der WNT-Proteine, die wiederum zur Gruppe der Wachstumsfaktoren gehören und aktiviert abhängig vom zellulären Kontext und Rezeptorstatus unterschiedliche Signalkaskaden. Einer seiner möglichen Rezeptoren ist ROR2, der zusammen mit ROR1 eine Subgruppe der großen Familie der Rezeptor-Tyrosinkinasen bildet. Die Interaktion von WNT5A und ROR2 führt zur Aktivierung des sogenannten nicht-kanonischen β-Catenin unabhängigen Signalweges. Dieser wiederum hemmt den kanonischen β-Catenin abhängigen Signalweg, der für Tumorprogress bekannt und im Pankreaskarzinom aktiviert ist. Die Rolle von WNT5A und ROR2 in der Karzinogenese verschiedener Tumorentitäten ist kontrovers. In manchen Tumoren wird ihnen eine Rolle als Tumorsuppressor zugeschrieben, in anderen besitzen sie ein onkogenetisches Potential. Die Rolle von WNT5A und ROR2 im duktalen Adenokarzinom des Pankreas ist insgesamt wenig erforscht, vor allem in Hinsicht auf ihre klinische Relevanz. Ziel dieser Arbeit war es, die Expressionsmuster von WNT5A und ROR2 im duktalen Adenokarzinom des Pankreas zu beschreiben, das Patientenkollektiv zu charakterisieren und Zusammenhänge mit klinisch pathologischen Parametern und dem Gesamtüberleben zu finden. Es standen 117 Tumorproben zur Verfügung, die im Zeitraum vom 2001 bis 2013 am Universitätsklinikum Leipzig chirurgisch gewonnen und im Institut für Pathologie des Klinikums aufbewahrt wurden. Das Patientenkollektiv umfasste 72 Männer und 45 Frauen mit einem medianen Alter von 66 Jahren. Die angefertigten Schnitte wurden immunhistochemisch mit Antikörpern gegen WNT5A und ROR2 charakterisiert. Im Anschluss daran beurteilten drei unabhängige Gutachter den prozentualen Anteil der Färbung im Schnitt und dessen Intensität in jeweils Tumor, Stroma und gesundem Gewebe. Daraus wurde ein Score berechnet, der Werte von 0¬–300 einnehmen konnte. Ausgehend von einem Cut-off-Wert wurden die Ergebnisse der mikroskopischen Auswertung in negative und positive Expression eingeteilt und eine statistische Analyse angeschlossen. Die WNT5A-Färbung zeigte eine starke positive Expression im Tumor und gesunden Gewebe, jedoch nur eine geringe Expression im Stroma. Dabei unterschied sich die WNT5A-Expression von Tumor zu Stroma sowie gesundem Gewebe zu Stroma signifikant. Eine ausgeprägte positive Expression von ROR2 war ebenso im Tumor und gesundem Gewebe zu verzeichnen, wohingegen der Rezeptor im Stroma nur gering exprimiert war. Die ROR2 Expression unterschied sich zwischen Tumor und Stroma und gesundem Gewebe und Stroma ebenfalls signifikant. Die Korrelationsanalyse der Expressionsmuster von WNT5A und ROR2 mit klinisch pathologischen Parametern zeigte keine Signifikanzen. Die Überlebenszeitanalyse in Abhängigkeit von klinisch-pathologischen Parametern ergab eine prognostische Relevanz für T- und N-Kategorie, UICC-Stadium, Grading, Lymphgefäß- und Veneninvasion sowie das Rezidivauftreten in Form von Metastasen. In der Multivarianzanalyse konnten der Befall von Lymphknoten (pN1) und ein Grading vom Grad 3 als ungünstige unabhängige prognostische Faktoren identifiziert werden. Die Überlebenszeitanalyse in Abhängigkeit der ROR2 und auch WNT5A-Expression in Tumor, Stroma und gesundem Gewebe zeigte keine Signifikanz. Die Ergebnisse dieser Arbeit zeigen insgesamt nur eine geringe klinische Relevanz von WNT5A, wohingegen in vitro-Untersuchungen ihm einen starken Einfluss in Hinsicht auf Migration, Proliferation und Invasionsverhalten zusprachen. ROR2 scheint vor allem im fortgeschrittenen Tumorstadium einen Einfluss auf das Überleben zu nehmen und durch eine positive Expression in Tumor und Stroma protektiv zu wirken. Denkbar wäre die durch seine Präsenz verstärkte Aktivierung des nicht-kanonischen Signalweges über WNT5A, die dann zur Hemmung des kanonischen Signalweges führen könnte. Um dieser Hypothese nachzugehen, sind weiterführende Untersuchungen unverzichtbar. Auch um die klinische Relevanz besser einschätzen zu können, sind größere Kollektive und umfassendere Analysen notwendig.

The exact mechanisms that regulate limb outgrowth the mouse embryo are unknown. Although there are several models, we favor a hypothesis where cells become polarized by signals secreted from the AER which orient their cell migration and/or divisions causing limb outgrowth. Clonal analysis has provided a mechanism to study cell behavior. We have generated a targeting construct containing the Fgf8 inhibitor, Sprouty2, in order to generate mutant clones for behavioral analyses in the limb. In order to more effectively study clonal behavior we report the modification of a novel clonal analysis approach, exo-utero surgery. We have modified, enhanced and proven that this technique can be used successfully in mouse embryos in which we directly apply 4-OHtamoxifen to the limb to induce YFP or β-gal reporter genes in limb mesenchyme. Using this method, we can closely control the timing and location of the induced clones and observe cell behavior during embryonic limb development. Phenotypes of Wnt5a-/- and Ror2-/- exhibit shortened limbs suggesting they function in a similar pathway. Wnt5a and Ror have been found to "colocalize" in the growing limb bud and have also been shown to bind in vitro. Here we show preliminary results about Wnt5a and Ror2 in vivo association by immunoprecipitation of limb bud extracts.

Despite over 60 years of study, the molecular pathways and mechanisms governing limb outgrowth and patterning remain poorly understood. Fgfs expressed in the AER are known to be necessary and sufficient for proximodistal limb outgrowth and have been proposed to have a chemoattractive role. Wnt5a is a secreted factor which is expressed in a gradient in the distal limb with the highest concentration next to the AER. The presence of the AER is necessary to establish this gradient. Expression of Wnt5a in a concentration dependant manner can be induced in the limb through the implantation of a bead soaked in recombinant Fgf4 protein. This indicates that Fgfs from the AER may establish the gradient of Wnt5a in the limb mesenchyme. Wnt5a-/- mutants exhibit severe shortening of the face, limbs, and body axis, with limbs being progressively truncated proximally to distally. In normal limb proximodistal outgrowth, cells are seen to grow directionally toward the AER. Previous studies done in the Barrow lab, as well as those done by myself, have shown that if a portion of the AER is removed and the cells proximal to this area are labeled, those which are close enough to intact AER will redirect their growth toward this intact AER. When Wnt5a secreting cells are implanted in the limb mesenchyme of the chick this ectopic source of Wnt5a is sufficient to redirect the growth of the mesenchyme cells toward the Wnt5a source. This indicates that the AER may mediate directed growth of limb mesenchyme cells through the establishment of the Wnt5a gradient which provides positional information to the cells. This Wnt5a gradient results in the recruitment of the mesenchyme cells toward the AER. The Ror2 receptor has been found to be involved in several different pathways involving Wnt5a which are involved in changes in polarity and migration. This makes Ror2 a likely candidate for causing changes in cell polarity and migration during distal outgrowth in the limb. To test whether Ror2 is necessary for the polarizing response of limb mesenchyme cells to the Wnt5a gradient in vivo I co-transfected a dominant-negative Ror2 (Ror2ΔC) and a GFP expression vector in the embryonic chick limb using sonoporation. Limb mesenchyme cells transfected with dominant-negative Ror2 grew as radial clones in contrast to the directional outgrowth of the control limb mesenchyme cells along the proximodistal axis. This indicates that cells expressing the dominant-negative Ror2 could no longer respond to the Wnt5a gradient in the limb mesenchyme. This supports a role for Ror2 as a receptor or co-receptor for Wnt5a in mediating directional growth and movement during proximodistal outgrowth and patterning in the limb.

Patients on peritoneal dialysis (PD) are reliant on the peritoneum to provide a semi-permeable barrier to allow for dialysis (solute clearance), salt and water removal (ultrafiltration). PD patients are at risk of developing peritoneal fibrosis and angiogenesis which can lead to a decline in peritoneal membrane function. Specifically, PD patients develop increased solute transport and decreased osmotic conductance leading to ultrafiltration failure. Peritoneal angiogenesis is the leading factor that results in augmented peritoneal membrane solute transport which is associated with worse outcomes – increased risk of mortality and PD technique failure. Transforming growth factor beta (TGFB) is one of the primary cytokines involved in inducing epithelial to mesenchymal transition (EMT) and fibrosis. We hypothesize that PD leads to injury of the epithelial lining of the peritoneum – the mesothelial cells. These cells undergo a transition process and transitioned mesothelium are a source for angiogenic and fibrogenic growth factors. Matrix Metalloproteinase (MMP) 9 is an angiogeneic factor and has been observed to correlate with increased expression of vascular endothelial growth factor (VEGF). MMP9 has the ability to cleave and activate membrane bound factors such as E-cadherin and b-catenin respectively. There is substantial evidence that the canonical WNT/b-catenin pathway is active during fibrosis, and angiogenesis in different biological contexts. Thus, we investigated the role of MMP9 and WNT signaling in peritoneal angiogenesis. Limited evidence exists describing the role of noncanonical WNT signaling but some reports suggest that non-canonical WNT signaling inhibits WNT/b-catenin signaling. Non-canonical WNT5A has differential effects based on receptor context and has been shown to block WNT/b-catenin signaling in the presence of Receptor Tyrosine Kinase Like Orphan Receptor 2 (Ror2). The overall hypothesis of this PhD thesis is that MMP9 and WNT signaling play a key role in inducing peritoneal angiogenesis and are associated with changes in peritoneal membrane function. We expect WNT5A and Ror2 to protect against peritoneal membrane injury. From the overnight effluent of stable PD patients, we cultured mesothelial cells and assayed these for expression of MMP and WNT related genes. MMP9 and WNT1 gene expression were observed to be strongly correlated with peritoneal membrane solute transport in patients on PD. WNT2 mRNA was also positively correlated with peritoneal solute transport. We overexpressed MMP9 in the mouse peritoneum to demonstrate its role in angiogenesis and confirmed these findings using MMP9 -/- mice. In addition to this, we have shown a novel mechanism by which MMP9 induces angiogenesis by E-cadherin cleavage and b-catenin mediated signaling. The observed cross-talk between MMP9 and b-catenin prompted investigation of the activation of canonical WNT/b-catenin signaling in development of peritoneal membrane injury. In an experimental model of TGFB induced pertioneal injury, we confirmed the activation of WNT/b-catenin signaling. In addition to this we, we blocked the WNT pathway and observed that WNT/b-catenin signaling is required to induce peritoneal angiogenesis. WNT5A mRNA was downregulated during TGFB induced injury suggesting a more protective role. Furthermore, several studies have demonstrated its ability to antagonize the WNT/b-catenin signaling pathway. We demonstrated that WNT5A protected against angiogenesis by blocking the canonical WNT pathway. WNT5A is thought to antagonize the WNT/b-catenin signaling pathway by signaling through receptor Ror2. In cell culture, we overexpressed TGFB and blocked Ror2. This resulted in elevated levels of VEGF and fibronectin suggesting that Ror2 is involved in mediating protection. Therefore, Ror2 possesses the ability to regulate VEGF and may be a potential candidate by which WNT5A mediates its protective effects. In conclusion, our findings identified MMP9 and WNT1 as potential biomarkers of increased peritoneal solute transport in patients that are on PD. We have also found a novel mechanism by which MMP9 interacts with b-catenin to induce peritoneal angiogenesis and have provided a first look at WNT/b-catenin signaling in peritoneal angiogenesis. Lastly, we have shown WNT5A to protect against peritoneal angiogenesis. Taken together, our findings are not only significant to the realm of PD research but hold wide applicability to research in the biomedical sciences. / Thesis / Doctor of Philosophy (PhD)

The AER has a vital role in directing embryonic limb development. Several models have been developed that attempt to explain how the AER directs limb development, but none of them are fully supported by existing data. I provide evidence that FGFs secreted from the AER induce a gradient of Wnt5a. I also demonstrate that limb mesenchyme grows toward increasing concentrations of Wnt5a. We hypothesize that the changing shape of the AER is critical for patterning the limb along the proximal to distal axis. To better understand the pathway through which Wnt5a elicits its effects, we have performed various genetic studies. We demonstrate that Wnt5a does not signal via the Wnt/β-catenin pathway. However, we show that Wnt5a mutants share many common defects with Vangl2 mutants suggesting that Wnt5a signals through the Wnt/planar cell polarity (PCP) pathway.