Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.

STK11/LKB1, a serine/threonine protein kinase, is a key upstream kinase of adenine monophosphate-activated protein kinase (AMPK), a necessary kinase in the control of metabolism for maintaining energy homeostasis. Although it has become clear that LKB1 is mutated in a significant number of Peutz¡VJeghers syndrome (PJS) and sporadic cancers, most frequently in adenocarcinoma of the lung, little is known about how the LKB1 signaling regulates the metabolic process and energy production underlying hypoxia and increased radiosensitivity of lung tumor. Here, we employed lung cancer cells as a model system to dissect the functional roles of LKB1 signaling in human lung adenocarcinoma. We found that LKB1 inhibits lung cancer cell migration, transformation and chemo-resistance in vitro after we restored LKB1 expression in LKB1 null A549 and H460 lung cancer cells. We also found that LKB1 prevents UV-induced DNA damage in human lung cancer cell lines by comet assay and activated UV-induced apopotsis by MTT assays. Furthermore, we designed a systems biology approach to provide a comprehensive protein-protein interaction analysis in order to elucidate the LKB1 tumor suppressor network in vivo. We employed Immunoprecipitation-HPLC- Mass Spectrometry (IP-LC-MS) to identify the novel proteins interacting with LKB1 under different cellular stress conditions. We have identified that LKB1 is involved in CFTR synthesis pathway underlying normoxia condition and participates in the glycolysis and gluconeogenesis pathways underlying hypoxia condition. Together, our findings indicated that LKB1 is involved in the regulation of cell migration, energy metabolism and DNA repair in lung cancer cells, and should provides insights to further exploit the concept of deranged cancer bioenergetics and aberrant growth signals to achieve more effective and selective strategies for lung cancer patients.

Wnt11 is a key signalling molecule that regulates cell polarity/migration during vertebrate development and also promotes the invasive behaviour of adult cancer cells. It is therefore essential to understand the mechanisms by which Wnt11 signalling regulates cell behaviour. The process of vertebrate gastrulation provides an excellent developmental system to study Wnt11 function in vivo. It is known that Wnt11 mediates coordinated cell migration during gastrulation via the non-canonical Wnt pathway that shares several components with a the planar cell polarity pathway (PCP) in Drosophila. However, the mechanisms by which these PCP components facilitate Wnt11 function in vertebrates is still unclear. While in Drosophila, the asymmetric localization of PCP components is crucial for the establishment of cell polarity, no asymmetric localization of Wnt11 pathway components have so far been observed in vertebrates. To shed light on the cellular and molecular mechanisms underlying Wnt11 signalling, I developed an assay to visualize Wnt11 activity in vivo using live imaging of Wnt11 pathway components tagged to fluorescent proteins. This allowed me to determine the sub-cellular distribution of these components and to correlate the effect of Wnt11 activity with the behaviour of living embryonic cells. I found that Wnt11 locally accumulates together with its receptor Frizzled7 (Fz7) at sites of cell-cell contacts and locally recruits the intra-cellular signalling mediator Dishevelled (Dsh) to those sites. Monitoring these apparent Wnt11 signalling centres through time-lapse confocal microscopy revealed, that Wnt11 activity locally increases the persistency of cell-cell contacts. In addition, I found that the atypical cadherin Flamingo (Fmi) is required for this process. Fmi accumulates together with Wnt11/Fz7 at sites of cell-cell contact and locally increased cell adhesion, via a mechanism that appears to be independent of known downstream effectors of Wnt11 signalling such as RhoA and Rok2. This study indicates that Wnt11 locally interacts with Fmi and Fz7 to control cell-contact persistency and to facilitate coherent and coordinated cell migration. This provides a novel mechanism of non-canonical Wnt signalling in mediating cell behaviour, which is likely relevant to other developmental systems. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 50 MB: Movies - Nutzung: Referat Informationsvermittlung der SLUB)

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and particularly common in China and Southeast Asia. It ranks the 2nd and 4th most common fatal cancer in males and females, respectively, in Hong Kong. Current treatments are not always effective, as recurrence and metastasis in HCC are difficult to tackle and the underlying mechanisms not fully understood. Aberration of Wnt signaling has been implicated in HCC; in this study, we investigated the underlying mechanisms of how aberrant Wnt signaling promoted HCC development. With Taqman Low Density Array (LDA) analysis on 38 pairs of HCC and the corresponding non-tumorous livers for 59 Wnt signaling related-genes, we found significant overexpression of the Wnt signaling intermediate, Dishevelled (Dvl)-3, in HCC (p = 0.014). This observation in LDA was confirmed in 36 additional HCC cases. Among a total of 74 cases studied, 28.38% showed more than 3-fold overexpression in the tumors as compared with the corresponding non-tumorous livers. Dvl3 overexpression positively correlated with the presence of venous invasion. We also observed significant correlation of Dvl3 expression with accumulation of β-catenin, a downstream effecter of Wnt/β-catenin signaling (p=0.028). We further characterized the functional roles of Dvl3 in contributing to the stem cell-like and metastatic properties of HCC. We found that Dvl3 knockdown in HCC cells suppressed cell proliferation, sphere formation, tumorigenicity in immunodeficient mice, chemo-resistance, and expression of stemness genes. We then examined whether Wnt/β-catenin was effectively modulated by Dvl3 and found that Dvl3 overexpression and knockdown, respectively, promoted and reduced the TOP/FOP luciferase reporter activity in HCC cells. This was accompanied by the expression of β-catenin target genes, EpCAM and LGR5, both of which are associated with HCC stemness. Furthermore, rescue with wild-type or constitutively active β-catenin partially restored the in vivo tumorigenicity suppressed by Dvl3 knockdown, indicating a partial role of β-catenin in mediating the effects of Dvl3 on HCC stemness. In addition, since cell migration is a critical determinant in metastasis, we assessed the HCC cell migratory ability in vitro using transwell migration assays and observed suppression of the cell migration ability upon Dvl3 knockdown. Also, the in vivo orthotopic model confirmed a role of Dvl3 in promoting metastasis, as stable Dvl3 knockdown in HCC cells resulted in a reduction in lung metastasis. Interestingly, the effect of Dvl3 on cell migration was independent of β-catenin, as knockdown of β-catenin had no effect on HCC cell migration in vitro. It was also not related to the phosphorylation of MYPT in Rho-ROCK signaling, which itself was previously implicated in HCC cells metastasis and reported as a downstream signaling of Dvl in development. In summary, our study has identified roles of Dvl3 in HCC stemness properties and cell migration and this may provide functional implication of Dvl3 overexpression, which significantly correlated with venous invasion in human HCCs. Also, β-catenin is partly responsible for the role of Dvl3 in HCC stemness but independent of that in cell migration. Functional characterization of Dvl3 in HCC may help future development of therapy targeting Dvl3 of Wnt signaling pathways. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

DKK1, SFRP1, WIF-1, and Wnt5a encode Wnt pathway genes that are frequently silenced by promoter hypermethylation in colorectal cancer. Despite attractive biological consequences of these events, it is unclear whether they contribute to patient prognostication or may influence tumour cell biology within distinct patient subsets. I sought to determine the prognostic roles of these methylation events in a large cohort of colorectal carcinomas from Ontario and Newfoundland. Methylation was quantified and associated with patient clinicopathlogical features. Methylation was present in cancer tissue. DKK1, Wnt5a, and SFRP1 were strongly and independently associated with tumour subtype in a manner that suggested subtype-specific activity of Wnt signaling. Methylation of DKK1 was a borderline prognosticator of favourable outcome. These results offer intriguing insight into subtype-specific biology and lead to a proposed model whereby methylation-induced Wnt bias may contribute to patient outcome.

Canonical Wnt signaling is involved in many aspects of development including axis specification and anterior-posterior neuroectoderm formation during vertebrate embryogenesis. Axin2, a homologue of Axin1, is thought to have a similar regulatory role within the cell, but differences in their expression and binding partners suggest Axin2 is not completely redundant with Axin1. To better understand Axin2 in canonical Wnt signaling, I utilized several approaches to explore its expression and function. In the zebrafish embryo, I found Axin2 is expressed in known active domains of Wnt signaling, suggesting an inducible regulatory role. Additionally, canonical Wnt signaling was sufficient and necessary to induce Axin2 expression and Axin2 was sufficient and necessary to inhibit Wnt signaling. As Wnt signaling is important in development and its dysregulation has been implicated in diseases such as colorectal cancer, this study helps advance our understanding of how Wnt signaling regulates itself through the use of negative feedback inhibitors, such as Axin2.

DKK1, SFRP1, WIF-1, and Wnt5a encode Wnt pathway genes that are frequently silenced by promoter hypermethylation in colorectal cancer. Despite attractive biological consequences of these events, it is unclear whether they contribute to patient prognostication or may influence tumour cell biology within distinct patient subsets. I sought to determine the prognostic roles of these methylation events in a large cohort of colorectal carcinomas from Ontario and Newfoundland. Methylation was quantified and associated with patient clinicopathlogical features. Methylation was present in cancer tissue. DKK1, Wnt5a, and SFRP1 were strongly and independently associated with tumour subtype in a manner that suggested subtype-specific activity of Wnt signaling. Methylation of DKK1 was a borderline prognosticator of favourable outcome. These results offer intriguing insight into subtype-specific biology and lead to a proposed model whereby methylation-induced Wnt bias may contribute to patient outcome.

Breast and pancreatic cancers are among the major causes of cancer mortality in our society. There has been a significant decline in mortality from breast cancer over the last two decades, while pancreatic cancer has an exceptionally poor prognosis. Although these malignancies have very different clinical outcomes they share the common feature that metastatic disease is almost uniformly fatal. The existence of cancer stem cells has been postulated as a major factor in tumour recurrence after traditional chemo- or radio-therapy. Addressing this important clinical question requires a deeper understanding of the biology of normal and cancer stem cells and the signalling pathways involved in their regulation. The identity of the pancreatic stem cell remains elusive. However, using a murine model of haematopoietic stem cell (HSC) transplantation I have demonstrated for the first time transdifferentiation of these bone marrow derived cells into mature pancreatic acinar cells, where they appear to contribute to cell turnover ultimately forming acini and lobules. These data show that HSC have surprising developmental plasticity and provide insight into a potential stem cell niche in the pancreas. The Hedgehog, Wnt and Notch signalling pathways play a critical role in early development and in the maintenance and self-renewal of stem cells. There is also increasing evidence that dysregulation of these pathways contributes to the development of many malignancies. There is relatively little information regarding their role in breast cancer development and progression. I used immunohistochemistry for key proteins in these pathways, sonic hedgehog, beta-catenin and Notch 1 in three substantial series of human breast lesions and determined that abnormal expression of these proteins is an early event in the development in breast cancer, and is associated with particular breast cancer subtypes, Shh and beta-catenin expression is associated predominantly with the basal-like phenotype and Notch 1 with the HER2 amplified phenotype. Overexpression of Shh in particular confers a worse clinical outcome in invasive ductal carcinoma. Furthermore, increased levels of Shh in a 3D culture model of non-transformed mammary epithelial cells resulted in disorganisation of acini and the development of an abnormal discohesive phenotype. Finally the role of Shh was investigated in a mammary epithelial transplantation model, where overexpression of Shh resulted in the development of hyperplasia of the mammary ductal epithelium. Together these data confirm that the Hedgehog, Wnt and Notch developmental pathways are dysregulated in breast cancer and represent viable targets for further investigation of potential novel therapies in breast cancer.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 55-69).