The high rate of obesity is having a significant impact on human health. Understanding the underlying biological mechanisms that regulate adipogenesis and adipocyte lipid metabolism is necessary to identify novel approaches that promote weight loss. Conjugated linoleic acid (CLA) is an example of a naturally-derived product reported to exhibit an anti-obesity effect. For this thesis, it was hypothesized that the anti-obesity effects of the t10-c12 CLA isomer is due to lipid droplet dynamics alteration through activation of the Wnt/β-catenin pathway, which leads to weight loss via affecting adipogenesis and/or adipocyte death. Testing of this hypothesis was achieved by examining the effects of the most biologically active CLA isomers, cis-9, trans-11 (c9-t11), trans-10, cis-12 (t10-c12) CLA using in vitro (3T3-L1 cell line) and in vivo (mouse) models. In 3T3-L1 preadipocytes, both c9-t11 and t10-c12 CLA stimulated early stage differentiation, while t10-c12 CLA inhibited late differentiation as indicated by fewer lipid droplets, lower adipokine levels, and decreased levels of perilipin-1 and phospho-perilipin-1 compared to null. The t10-c12 CLA isomer decreased hormone-sensitive lipase (HSL) levels and inhibited lipolysis by activating protein kinase Cα (PKCα). As well, t10-c12-CLA inhibited adipocyte differentiation by stabilizing β-catenin, which sequesters peroxisome proliferator-activated receptor-γ in an inactive complex. Reduced body weight in both db/db and C57B/L6 mice fed t10-c12 CLA was due to less white and brown fat mass without changes in lean body mass or an alteration in feed intake compared to their respective control. t10-c12 CLA did not stimulate cell death in white adipose tissue. Immune cell infiltration was decreased in calorie restricted pair weight control mice, but not with CLA. t10-c12 CLA-induced weight loss did not improve hyperglycemia in db/db mice. In conclusion, the anti-adipogenic effects of t10-c12 CLA in vitro result from stabilization of β-catenin, which alters lipid droplet dynamics through HSL levels and perilipin-1 phosphorylation via the activation of PKCα. In contrast, t10-c12 CLA promotes loss of adipose tissue in vivo, possibly by activating β-catenin, but without influencing either adipogenesis or adipocyte clearance. This study suggests a novel mechanism for the anti-obesity effect of t10-c12 CLA, and highlights the possible side-effects associated with t10-c12 CLA consumption. / February 2016

Beim humanen nichtkleinzelligen Bronchialkarzinom ist die schrittweise Progression vom gutartigen Tumor zur malignen Metastasierung weitestgehend ungeklärt. In einem transgenen Mausmodell für das humane nichtkleinzellige Bronchialkarzinom, in dem in Lungenepithelzellen eine onkogene Mutante der Proteinkinase C-RAF exprimiert wird, können einzelne Schritte im Prozess der malignen Progression entschlüsselt werden. Die durch C-RAF induzierten Adenome zeichnen sich durch eine hohe genomische Stabilität in den Tumorzellen, durch starke interzelluläre Adhäsionskontakte zwischen den Tumorzellen und das Fehlen einer malignen Progression aus. Hier wurde demzufolge untersucht, ob die Auflösung der E-Cadherin-vermittelten Zellkontakte zwischen den einzelnen Tumorzellen eine Metastasierung auslösen könnte. Es wurden zwei genetische Ansätze verfolgt, um die Rolle der Tumorzelladhäsion im C-RAF Modell zu bewerten, die konditionelle Eliminierung des E-Cadheringens Cdh1 sowie die regulierbare transgene Expression von dominant-negativem E-Cadherin. Die Auflösung der E-Cadherin-vermittelten Zelladhäsion führte zur Neubildung von Tumorgefäßen, welche in der frühen Phase der Gefäßbildung durch Wiederherstellung des Zellkontakts reversibel war. Die vaskularisierten Tumore wuchsen schneller, bildeten invasive Fronten aus und führten zur Ausbildung von Mikrometastasen. Es konnte gezeigt werden, dass Beta-Catenin für die Induktion der Angiogenesefaktoren VEGF-A und VEGF-C in Lungentumorzellinien des Menschen und der Maus essentiell war. Lungentumorzellen aus den in situ Tumoren mit aufgelösten E-Cadherin-vermittelten Zellkontakten exprimierten Gene endodermaler und anderer Zellabstammung, was epigenetische Reprogrammierung in Tumorzellen als den Mechanismus bei der malignen Progression vermuten lässt. / Steps involved in the progression of non-small cell lung cancer (NSCLC) to metastasis are poorly understood. Expression of oncogenic C-RAF in lung epithelial cells has yielded a model for non-small cell lung cancer (NSCLC). The induced adenomas are characterised by high genomic stability, a lack of tumor progression and pronounced cell-cell contacts raising the question whether disruption of E-cadherin complexes would promote progression to metastasis. Two genetic approaches were used to evaluate the role of adherens junctions in a C-RAF driven mouse model for NSCLC: conditional ablation of the Cdh1 gene and expression of dominant negative (dn) E-cadherin. Disruption of E-cadherin function caused massive formation of intratumoral vessels that was reversible in the early phase of induction. Vascularized tumors grew more rapidly, developed invasive fronts and gave rise to micrometastasis. ß-catenin was identified as a critical effector of E-cadherin disruption leading to up-regulation of angiogenic inducers (VEGF-A and VEGF-C) in mouse and human lung tumor cell lines. In vivo, lung tumor cells with disrupted E-cadherin expressed ß-catenin target genes of endodermal and other lineages suggesting that reprogramming may be involved in metastatic progression.

Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1. We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. Without STAT3, the brown preadipocytes have increased apoptosis early in the terminal differentiation phase. We also show that the block in differentiation is caused by an inability of STAT3 knockouts to down regulate β-catenin by the end of the induction phase. Application of Wnt/β-catenin inhibitors or knockdown of β-catenin during the induction phase is sufficient to fully rescue differentiation of brown adipocytes from the Myf5+ lineage, including reduction in apoptosis, restoration of histone acetylation in the UCP1 promoter and enhancer regions, and full restoration of the expression of brown fat genes. Finally, we show that in the beige lineage, STAT3 is also necessary during the induction phase and can be rescued by Wnt/β-catenin inhibitors, although the rescue is not as robust as it is in the Myf5+ lineage.

Wnt/β-catenin signaling and asymmetric cell division are essential to development and homeostasis in metazoans; these two mechanisms join into one in the Wnt/β-catenin Asymmetry (WβA) pathway in the nematode C. elegans. In WβA, nuclear asymmetry of two β-catenins, SYS-1 and WRM-1, is achieved by two parallel pathways that reduce SYS-1 and WRM-1 levels in the anterior daughter and increase their levels in the posterior daughter. While it is known that many conserved regulators of Wnt signaling are involved in WβA, how these components interact to achieve SYS-1 and WRM-1 asymmetry is not well understood. In this thesis, genetics, transgenics, and live-imaging are used to demonstrate how WβA regulates it’s multiple outputs. It is shown that APR-1/APC and PRY-1/Axin control asymmetric localization of both SYS-1 and WRM-1, and that Wnt signaling explicitly controls APR-1 regulation of either β-catenin via the kinase KIN-19/CKIα. Additionally, it is demonstrated that the Dishevelled proteins DSH-2 and MIG-5 are positive regulators of SYS-1, but negative regulators of WRM-1. Additionally, data from a screen designed to identify novel kinase regulators of Wnt signaling/asymmetric cell division is presented. Overall, this thesis takes current knowledge of conserved Wnt signaling component function and provides a compelling model of how those components are adapted to asymmetric cell division.

Type II diabetes and hyperinsulinemia are associated with increased risks of developing colorectal cancer (CRC). Detailed mechanisms underlying this correlation, however, are yet to be explored. The present study demonstrates that insulin increases the expression of proto-oncogenes c-Myc and cyclin D1 via both translational and transcriptional mechanisms. We show here that insulin stimulates c-Myc gene translation via an Akt/PKB-dependent mechanism involving the mTOR signaling pathway. More importantly, we show for the first time that transcriptional stimulation of c-Myc and cyclin D1 expression by insulin involves a novel Akt/PKB-independent signal crosstalk between insulin and canonical Wnt signaling pathways. We then identified p21-activated protein kianse 1 (PAK-1) as a novel mediator for insulin and Wnt/beta-catenin (b-cat) molecular crosstalk, involving MEK/ERK signaling. Furthermore, we found that insulin treatment leads to increased b-cat phosphorylation at Ser675, and this is associated with increased b-cat nuclear content and increased b-cat interaction with Tcf/Lef-binding elements (TBEs) of the human c-Myc gene promoter. Lastly, we demonstrated that insulin signaling directly alters the expression levels of components of the Wnt signaling pathway, including fizzled homology 4 (Fdz-4) and TCF7L2 (=TCF-4). This study not only demonstrated the existence of signaling crosstalk between insulin and canonical Wnt signaling pathways at multiple levels, it reveals molecular mechanisms for observed correlation between CRC and hyperinsulinemia. The growing evidence implicating PAK-1 in various human tumorigenesis has emerged PAK-1 as a potential therapeutic target. Our discovery of PAK-1 functioning as a novel central mediator for insulin and Wnt signaling crosstalk in intestinal cells suggests that PAK-1 may potentially be a good target candidate for treating patients with CRC, especially those who have Type II diabetes or experience hyperinsulinemia.

Aggressive fibromatosis is a fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in FAP patients. Tumours are characterized by the stabilization of beta-catenin. Current therapies have yet to offer complete success for primary and recurrent tumours, and there remains a need for more effective therapeutic strategies. In this work, we demonstrate the anti-neoplastic and beta-catenin modulating capacities of Nefopam, a currently approved analgesic agent. We found that Nefopam was able to decrease cell viability and proliferation as well as total beta-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, Nefopam reduced the number of tumours formed in the Apc+/Apc1638N aggressive fibromatosis mouse model. We also demonstrated that androgens contribute to the development of tumours and could also modulate beta-catenin levels as indicated in Testosterone-treated orchidectomized Apc+/Apc1638N mice. Together, this work suggests that Nefopam and androgen signaling-blocking agents are potential candidates to effectively manage aggressive fibromatosis.

Heart valve interstitial cells (VICs) undergo activation and proliferation in repair and disease, but the mechanisms are not fully understood. We hypothesize that the establishment of N-cadherin/β-catenin cell-cell contacts may decrease VIC activation, and that Wnt3a/β-catenin signaling may increase VIC proliferation. VIC cultures of different densities are stained for α-SMA, cofilin, TGF-β, pSmad2/3, N-cadherin and β-catenin, and probed for phospho-β-catenin by Western blot. Low density VIC cultures are treated with exogenous Wnt3a and measured for cell number, proliferation, apoptosis, α-SMA, β-catenin, and β-catenin-mediated transcription. β-Catenin siRNA knockdown is used to assess β-catenin specificity. Increased staining of α-SMA, cofilin, TGF-β, pSmad2/3, nuclear β-catenin, and increased phospho-β-catenin are associated with few cell-cell contacts. Wnt3a increased VIC cell number, proliferation, nuclear β-catenin and β-catenin-mediated transcription without affecting activation and apoptosis, and proliferation is abolished by β-catenin siRNA. Thus, N-cadherin/β-catenin cell-cell contacts may inhibit VIC activation and Wnt3a/β-catenin signaling may increase VIC proliferation.

Aggressive fibromatosis is a fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in FAP patients. Tumours are characterized by the stabilization of beta-catenin. Current therapies have yet to offer complete success for primary and recurrent tumours, and there remains a need for more effective therapeutic strategies. In this work, we demonstrate the anti-neoplastic and beta-catenin modulating capacities of Nefopam, a currently approved analgesic agent. We found that Nefopam was able to decrease cell viability and proliferation as well as total beta-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, Nefopam reduced the number of tumours formed in the Apc+/Apc1638N aggressive fibromatosis mouse model. We also demonstrated that androgens contribute to the development of tumours and could also modulate beta-catenin levels as indicated in Testosterone-treated orchidectomized Apc+/Apc1638N mice. Together, this work suggests that Nefopam and androgen signaling-blocking agents are potential candidates to effectively manage aggressive fibromatosis.

Heart valve interstitial cells (VICs) undergo activation and proliferation in repair and disease, but the mechanisms are not fully understood. We hypothesize that the establishment of N-cadherin/β-catenin cell-cell contacts may decrease VIC activation, and that Wnt3a/β-catenin signaling may increase VIC proliferation. VIC cultures of different densities are stained for α-SMA, cofilin, TGF-β, pSmad2/3, N-cadherin and β-catenin, and probed for phospho-β-catenin by Western blot. Low density VIC cultures are treated with exogenous Wnt3a and measured for cell number, proliferation, apoptosis, α-SMA, β-catenin, and β-catenin-mediated transcription. β-Catenin siRNA knockdown is used to assess β-catenin specificity. Increased staining of α-SMA, cofilin, TGF-β, pSmad2/3, nuclear β-catenin, and increased phospho-β-catenin are associated with few cell-cell contacts. Wnt3a increased VIC cell number, proliferation, nuclear β-catenin and β-catenin-mediated transcription without affecting activation and apoptosis, and proliferation is abolished by β-catenin siRNA. Thus, N-cadherin/β-catenin cell-cell contacts may inhibit VIC activation and Wnt3a/β-catenin signaling may increase VIC proliferation.

Type II diabetes and hyperinsulinemia are associated with increased risks of developing colorectal cancer (CRC). Detailed mechanisms underlying this correlation, however, are yet to be explored. The present study demonstrates that insulin increases the expression of proto-oncogenes c-Myc and cyclin D1 via both translational and transcriptional mechanisms. We show here that insulin stimulates c-Myc gene translation via an Akt/PKB-dependent mechanism involving the mTOR signaling pathway. More importantly, we show for the first time that transcriptional stimulation of c-Myc and cyclin D1 expression by insulin involves a novel Akt/PKB-independent signal crosstalk between insulin and canonical Wnt signaling pathways. We then identified p21-activated protein kianse 1 (PAK-1) as a novel mediator for insulin and Wnt/beta-catenin (b-cat) molecular crosstalk, involving MEK/ERK signaling. Furthermore, we found that insulin treatment leads to increased b-cat phosphorylation at Ser675, and this is associated with increased b-cat nuclear content and increased b-cat interaction with Tcf/Lef-binding elements (TBEs) of the human c-Myc gene promoter. Lastly, we demonstrated that insulin signaling directly alters the expression levels of components of the Wnt signaling pathway, including fizzled homology 4 (Fdz-4) and TCF7L2 (=TCF-4). This study not only demonstrated the existence of signaling crosstalk between insulin and canonical Wnt signaling pathways at multiple levels, it reveals molecular mechanisms for observed correlation between CRC and hyperinsulinemia. The growing evidence implicating PAK-1 in various human tumorigenesis has emerged PAK-1 as a potential therapeutic target. Our discovery of PAK-1 functioning as a novel central mediator for insulin and Wnt signaling crosstalk in intestinal cells suggests that PAK-1 may potentially be a good target candidate for treating patients with CRC, especially those who have Type II diabetes or experience hyperinsulinemia.