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.

The Wnt pathway is a fundamental signalling pathway conserved in all animals, regulating growth, differentiation, embryonic development, and tissue homeostasis in adults. Wnt signalling is kept quiescent by ubiquitin-mediated degradation of the transcription factor β-catenin, orchestrated by a group of proteins called the Destruction Complex. Aberrant Destruction Complex activity is a common theme in many cancers, and is the primary cause of colon cancer. Through mass spectrometry analysis of Axin protein complexes (a key Destruction Complex component) we identified the deubiquitinating enzyme USP34 as an Axin-interacting protein. Functional studies showed USP34 functions to positively regulate Wnt signalling, acting downstream of β-catenin stabilization. While characterizing USP34 we also discovered a new positive regulatory role for Axin in promoting signalling that is dependent on its nuclear localization. Our results suggest that USP34 stabilizes the nuclear pool of Axin through regulating its ubiquitination and offers a potential strategy to target pathological Wnt signalling.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.

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.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.

<p>The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.</p><p>In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury. </p><p>Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.</p> / Dissertation

The Wnt signaling pathway is highly conserved and participates in many important cellular functions including differentiation, embryonic development and tissue generations. £]-catenin, the central mediator of the Wnt signaling, interacts with the TCF/LEF family of transcription factors in the nucleus and initiates downstream gene transcription. In addition, £]-catenin is known as a proto-oncogene implicated in numerous cancers including colorectal, cervical, endometrial and skin cancer. Chibby (Cby) is evolutionarily conserved in many species and acts as a repressor of Wnt/£]-catenin signaling. In our previous study, we have established that Cby over-expression attenuated £]-catenin translocation to nucleus and its transcriptional activity. Thus, it was hypothesized that Cby may possess potential tumor suppressing capabilities. In the present study, we first explored endogenous Cby expression status in human cervical cancer cells: HeLa and SiHa cell lines. It was observed that Cby mRNA and protein levels were significantly down-regulated in both cancer lines compared with primary cervical cells. We then conducted functional assays of tumorigenicity on both cells using adenoviral-encoded Cby and its NLS (nuclear localization signaling) deleted variant (Cby∆NLS). It was found that gene delivery of Cby or Cby∆NLS inhibited the proliferation, invasiveness, and colony forming in HeLa and SiHa cells. Immunofluorescent analysis revealed that Cby or Cby∆NLS gene transfer reduced the expression of Ki-67, a cell proliferative marker. Furthermore, Cby or Cby∆NLS restoration induced apoptosis and perturbed cell cycle progression in both cervical cancer cells. Finally, Cby over-expression decreases the expression of £]-catenin/TCF4 regulated genes such as c-myc and PCNA, which might contributed to the anti-neoplastic mechanism for Cby in cervical cancer cell lines. Our results strongly suggest that Cby may serve as a tumor suppressor gene during cervical carcinogenesis, and may facilitate in creation of new therapeutic methods.

Leukocyte cell-derived chemotaxin 2 (LECT2) is first isolated as a 16-kDa secreted protein from cultured fluid of phytohemagglutinin-activated human T-cell leukemia SKW-3 cells. Recently LECT2 has shown to be synthesized by human hepatocytes and stimulates the growth of chondrocytes. LECT2 is involved in chemotactic factor to neutrophils and may be associated with rheumatoid arthritis. Besides, LECT2 is evolutionarily conserved and acts as a repressor in the Wnt/£]-catenin signaling pathway. Wnt/£]-catenin signaling is implicated in liver carcinogenesis. However, the exact roles of LECT2 in liver carcinogenesis are not yet well characterized. This study is to investigate the extra roles of LECT2 in Wnt signaling. Our results showed that adenoviral administration of LECT2 over-expression suppress oncogenic processes such as migration, invasion, proliferation and colony formation, as well as alteration in cell cycle distributions. In animal model significantly suppress liver malignancies in orthotopic Novikoff hepatoma. In conclusion, we show that ad-LECT2 gene delivery attenuated cell carcinogenesis process via downregulated Wnt/£]-catenin signaling in vitro and suppressed tumor growth in vivo. Besides LECT2 over-expression represents a novel therapeutically factor for hepatocelluar carcinoma.

Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.

<p>Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, we aimed to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition.</p><p>First, in a microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PF, we observed alterations of several Wnt pathway genes, including the Wnt inhibitor Secreted Frizzled Related Protein 3 (SFRP3). Loss-of-function studies interrogated the role of SFRP3 in human aRMS cell lines using shRNAs. Suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. SFRP3 suppression modestly increased Wnt signaling; however, activation of the Wnt pathway in human aRMS cells in vitro and in a xenograft murine model of aRMS in vivo only partially recapitulated the phenotype observed with SFRP3 suppression. To identify other signaling pathways downstream of SFRP3 signaling, we conducted an oncogenic signaling pathways screen and a microarray. In the former, we identified Notch signaling as conferring resistance to SFRP3 suppression-mediated decreased cell growth and confirmed Notch crosstalk with Wnt signaling and SFRP3 in aRMS cells. In the latter, SFRP3 suppression increased genes associated with skeletal muscle differentiation and decreased those associated with cell cycle progression. </p><p>Second, we established a role for SFRP3 in a conditional xenograft murine model of aRMS. Doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold. Analysis of the tumors by qPCR and IHC revealed an increase in myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. In conclusion, SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.</p> / Dissertation