▼ The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possesses unique self-renewal, expansion and differentiation activities in tumorigenesis. While this theory remains controversial in solid tumor studies, the enriched cancer stem cell population has been characterized by drug-resistant side population (SP), CD133pos, ALDHhigh and several other markers in many solid tumors. Using human lung cancer cell lines (A549 and H441) and primary human non-small cell lung adenocarcinoma (NSCLA) cells, we demonstrated that SP cells isolated from NSCLAs have significantly increased migration, invasion and metastatic activities compared to non-SP cells. We show that Rac1, a member of Rho-GTPases, is a critical mediator of the NSCLA SP CSC activity, by regulating SP cell adhesion, migration, invasion, and lung colonization activities. We further established that NSCLAs SP cells represent a distinct population from phenotypic CSC populations marked by CD133pos, ALDHhigh, or CD24lowCD44high, with each displaying enriched CSC activity demonstrated by expression of self-renewal genes and tumor-initiating activity in xenografted mice. Non-CSCs and CSCs defined by either absence or presence of single CSC marker respectively (SP, ALDHhigh, CD133pos, and CD24lowCD44high) are plastic and dynamic, with a distinct inter-conversion kinetics. Epithelial-mesenchymal transition (EMT) previously proposed as one of the underlying mechanisms of CSC plasticity, abolishes NSCLAs SP cells while increasing other CSC markers such as ALDHhigh, CD133pos and CD24lowCD44high. Rac1 activity was significantly increased in cells that have undergone EMT. Inhibition of Rac1 activity by NSC23766, a Rac1 inhibitor, or Rac1 knockdown, partially blocks EMT process and suppresses EMT induced phenotypic non-CSC to CSC conversion. Thus, we propose that there exist multiple, phenotypically distinct cancer cell subpopulations with CSC properties in NSCLA cells and phenotypic non-CSCs and CSCs can be dynamically regulated through EMT. Importantly, targeting Rac1 GTPase that is intimately involved in EMT process may represent a novel strategy in inhibiting CSC activity and suppressing non-CSC to CSC transition. Advisors/Committee Members: Zheng, Yi.

▼ Cancer remains one of the scourges of modern life despite our radical advances in medical technology and treatment. The need for improved therapies is apparent in the pediatric sarcoma population, including patients with rhabdomyosarcoma, who have a five-year survival of less than thirty-percent when diagnosed with metastatic disease. One emerging anti-cancer approach is the use of oncolytic viruses. Viruses derived from wild-type herpes, but possessing genetic modifications to alter their virulence and render them safer, have been tested in clinical trials and show promise as an anti-cancer treatment. The use of any oncolytic agent is likely impacted by the functions of the patient’s immune system; in theory, the host could mount a beneficial attack against the cancerous cells, or a harmful attack against the treatment agent itself, the latter of which would be expected to limit the anti-cancer activity of oncolytic viruses. I sought to develop a suitable model for exploring the interplay between the tolerogenic regulatory T-cells and the anti-viral and anti-tumor immune effector cells, which could be used to determine their role in the presence of oncolytic virus treatment. To this end, I required a tumor model in animals which possess an intact adaptive immunity for this study, yet human HSV replication is generally attenuated in mouse cells, in contrast to human tumor cells. Thus, I first screened mouse tumors for susceptibility to HSV infection. I used a mouse genetically susceptible to rhabdomyosarcoma due to expression of the hepatocyte growth factor/scatter factor transgene in the INK4a/ARF-/- background. I tested several cell lines derived from tumors to determine their ability to be killed by and support the replication of an oncolytic herpes simplex virus, rRp450. I also tested their tumorigenicity in syngeneic mice and response to intratumoral virus injection. To determine the role of regulatory T-cells in the anti-tumor immune response I used a Foxp3-DTR transgenic model which expresses the diphtheria toxin receptor under control of the Foxp3 (T-reg selective) promoter. I, along with my collaborators, also established a method to determine the extent of the anti-viral immune response using an immune tetramer. This tetramer incorporates the C57BL/6 MHC class I molecule (H2-Kb haplotype) and the immuno-dominant glycoprotein B peptide fragment (gB498-505) and allows for direct quantitation of the anti-viral cytotoxic T lymphocyte response. Several explanted tumor cell lines were found to support oncolytic virus replication, but only one was found to be tumorigenic in C57BL/6 mice. The Foxp3-DTR animals were found to be quite effective at depleting regulatory T-cells and possess many advantages over traditional antibody-mediated depletion strategies. Finally, the immune tetramer was found to be capable of evaluating the anti-viral immune response, as tetramer-staining was significantly increased in animals pre-treated with HSV versus mock-injected naïve controls. Thus, I have established an explantable mouse model of rhabdomyosarcoma that, similar to human rhabdomyosarcoma cells, is susceptible to oncolytic HSV infection. This model should prove ideal to study the role of regulatory T-cells in regulating the anti-tumor and anti-viral immune responses during oncolytic virotherapy. Advisors/Committee Members: Hildeman, David.

▼ Mutations in ZIC3 result in X-linked heterotaxy in humans, a syndrome consisting mainly of left-right (L-R) patterning defects, midline abnormalities, and cardiac malformations. Zic3 is highly expressed during gastrulation in the neuroectoderm and mesoderm of mouse and Xenopus laevis suggesting a conserved functional role in early development. To further investigate the mechanistic role of Zic3, loss of function analysis using morpholino antisense oligonucleotides was performed in Xenopus laevis. In Xenopus, knockdown of Zic3 expression results in randomization of Pitx2, a molecular marker of L-R patterning, at stage 23. At later stages, gut coiling and heart looping are reversed or ambiguous in 60% of morphants. These data recapitulate and extend phenotype information on loss of function in humans and mouse and demonstrate that the function of Zic3 in L-R patterning is conserved across species. Although loss of function of Zic3 in mouse results in abnormal L-R patterning and cardiac malformations, Zic3 null mice also exhibit defects in gastrulation, neural tube closure, and axial patterning, suggesting an earlier role in morphogenesis. These patterning events share a requirement for proper convergent extension (C-E) morphogenesis. Assay of characteristic C-E events in Xenopus, including blastopore closure, notochord elongation, and anterior-posterior axis length, demonstrates significant impairment in Zic3 morphants as compared to control morphants. Taken together, these results indicate that Zic3 plays a previously unrecognized role during early vertebrate development suggesting that early convergent extension defects underlie left-right asymmetry patterning defects at later stages Advisors/Committee Members: Ware, Stephanie.

▼ The lymphatic vascular system is critical for several key physiological functions including maintenance of fluid homeostasis by absorbing water and macromolecules from the interstitium, uptake of dietary lipids and fat soluble vitamins, and trafficking of immune cells. There are several diseases in which the lymphatic system is involved. Malformation or lack of lymphatic vasculature causes lymphedema, a debilitating and disfiguring disease that dramatically decreases quality of life. Overproduction of lymphatic vessels can lead to formation of lymphatic malformations. Other diseases associated with the lymphatic system include trafficking and migration of cancer cells and also its role in altered lipid transport, which is associated with obesity and aggravation of inflammatory bowel disorders such as Crohn’s Disease. Although there are many diseases associated with the lymphatic system, with results ranging from severely diminishing quality of life to death, comprehensive understanding of these diseases and cures for them remains elusive. Development of curative treatments for these diseases will first require a better scientific understanding of the patterns and processes of lymphatic development. The goal of this work is to further the field of developmental lymphangiogenesis by first describing the spatial and temporal emergence of developing lymphatic vessels in the zebrafish model organism. Zebrafish are particularly amendable to studies of developmental lymphangiogenesis due to their ex utero development, allowing direct optical access to nascent lymphatic vessels as they form within the living embryo. The anatomical mapping of zebrafish lymphatics will facilitate its use by researchers as we iv continue to make strides in this poorly understood area of vascular research. The anatomical mapping also led to the discovery of the supra-intestinal lymphatic vessel (SIL), which has the potential to open new avenues for understanding how dietary lipids are absorbed and may have implications that the zebrafish could be utilized to study the connection of the lymphatic system in lipid uptake and obesity. Once the spatiotemporal anatomy of the zebrafish was mapped, we began to explore the role of extracellular fluid on the emergence and development of this vascular network. We discovered normal interstitial flow patterns are critical for proper lymphatic vascular development. Experimentally increasing or decreasing the velocity of interstitial flow, via alterations in blood pressure, ablates the normal formation of the thoracic duct (the primary lymphatic vessel in the zebrafish). The loss of lymphatic vessel development under conditions of both increased and decreased hydrodynamic forces suggest that fluid flow may be helping to establish and maintain an appropriate biomorphological gradient which serves as a migratory cue for the LEC translocation from the parachordal vessel to the site of eventual coalescence and vessel assembly. While our collective understanding of lymphatic development is far from comprehensive, it is our hope that this work will aid in furthering the field to ultimately find cures for those that suffer from lymphatic system disease. Advisors/Committee Members: Hove, Jay.

▼ SAM Pointed Domain Containing ETS Transcription Factor (SPDEF) is a new member of ETS transcription factor family that is originally discovered in the epithelium of prostate. Like other ETS family members, SPDEF is involved in multiple biological processes including cell fate determination and specification, cell proliferation, epithelial-to-mesenchymal transformation and migration. Although much work has been done to elucidate the role of SPDEF in normal physiological conditions and in cancers, its expression level in cancer is still controversial. Similarly, there is no conclusive message on the role of SPDEF in cancer despite the fact that several in vitro studies have been done. We first overviewed the discovery and properties of SPDEF, followed by a comprehensive review of the role of SPDEF, with a special focus on its role in the development of cancer. In the second chapter, we utilized a xenograft model to manipulate SPDEF level in mouse prostate cancer. After orthotopic injection, we found that overexpression of SPDEF resulted in smaller tumors in mice. We also showed that several target genes related to cell proliferation and migration are down-regulated by SPDEF overexpression. We confirmed down-regulation of these genes in prostate cancer cell lines. In fact, we found that SPDEF overexpression inhibited cell migration, resulting in reduced aggressiveness of tumor cells. In addition, SPDEF overexpression was shown to suppress tumor growth via alteration of cell cycle profile. Collectively, these data indicate that SPDEF suppresses tumor growth via inhibition of cell proliferation and migration. Advisors/Committee Members: Kalin, Tanya.

▼ The Hox family of transcription factors control differential gene expression in specific cells and tissues along the anterior-posterior (A-P) axis of organisms from worms to humans, yet how they achieve this function remains poorly understood. The work presented in this thesis uses a combination of molecular biological approaches including transgenic reporter assays and genetics together with biochemical approaches including electrophoretic mobility shift assays and coimmunoprecipitation experiments to investigate the mechanisms regulating gene expression by Hox factors in specific cell types during the development of the model organism, Drosophila melanogaster. I have identified a cis-regulatory module (CRM) of the gene rhomboid (rho), which is a serine protease that serves as the rate limiting step in sending an epidermal growth factor (EGF) signal in Drosophila. This CRM recapitulates rho expression in a subset of sensory organ precursor cells and can functionally rescue a rho loss of function phenotype of these cells. I demonstrate that this CRM is regulated by Hox factors using multiple mechanisms to integrate cell-type restricted transcription factors and achieve specific activation within the proper cell type. First, I demonstrate a novel, conserved mechanism of Hox action whereby differential gene expression is achieved via competition for DNA binding to overlapping binding sites of the Growth Factor Independence family of transcriptional repressors and common, cooperatively-binding complexes formed by Hox proteins and their cofactors. Moreover, I found that distinct Hox factors form complexes that have differing affinities for the conserved binding site on the rho CRM, which affects their ability to compete for DNA binding. Thus, differences in affinity for DNA binding of distinct Hox/cofactor complexes contribute to differential gene expression. Next, I found that Hox factors gain functional specificity by differentially interacting with the Drosophila Pax2 (DPax2) ortholog on the rho CRM. Specifically, I show that an abdominal Hox factor can physically interact with Pax2 while a thoracic Hox factor cannot. Additionally, I show that these two Hox factors differ in their ability to interact with common cofactors of the Pbx and Meis families in the presence of DPax2. As robust activity of the rho CRM and the rho gene in these cells depends on Hox, Hox cofactor, and Pax inputs, the data characterize a mechanism used by Hox factors to differentially regulate rho in specific cell types. Finally, since each of these factors is important in both development and disease and highly conserved throughout the animal kingdom, the mechanisms demonstrated by this work are likely to be broadly applicable to a variety of developmental processes and beneficial to the study of human disease. Advisors/Committee Members: Gebelein, Brian.

▼ Allergic inflammation affects hundreds of millions of people worldwide. The pathogenesis of allergic inflammation is an area under active investigation. However, the role of miRNAs, a novel class of small RNA molecules 19-25 base pairs long that regulates gene expression post-transcriptionally, had not been explored at the start of this thesis. Using asthma and eosinophilic esophagitis (EoE) as model diseases of allergic inflammation, we identified miRNA signatures from murine experimental asthma, human EoE esophageal biopsies and IL-13 stimulated human esophageal / human bronchial epithelial cells. Accordingly, we identified commonly regulated miRNAs such as miR-21 and miR-146 that were involved in multiple allergic responses as well as miRNAs that were directly regulated by IL-13 including miR-375. Focusing on one of the most up-regulated miRNA miR-21, we developed a series of findings demonstrating that it directly represses IL-12p35 expression. Notably, miR-21 had the highest expression in dendritic cells, and miR-21 deficient dendritic cells produced significantly more IL-12 in response to LPS stimulation. Furthermore, miR-21 deficient mice had reduced lung eosinophilia following induction of allergic inflammation, with a prominent up-regulation of IL-12/IFNγ related pathways in the lung. Bronchoalveolar lavage fluid (BALF) from allergen challenged miR-21 deficient mice had increased Th1 cytokine IFNγ and decreased Th2 cytokine IL-4 compared to wildtype littermates. Re-stimulation of CD4+ T helper cells from the mice showed similar results. Conversely, miR-21 deficiency significantly enhanced the Th1 associated cutaneous delayed-type hypersensitivity responses. In complementary experiments concerning the role of miRNAs in allergy related responses, we found up-regulation of both miR-21 and miR-223 in an ex vivo eosinophil differentiation model. Notably, miR-21 deficiency led to reduced eosinophil progenitor growth while miR-223 deficiency enhanced eosinophil growth. Finally, in a distinct set of experiments aimed to define the role of miRNAs in allergic responses induced directly by IL-13, we noted that IL-13 stimulation of lung and esophageal epithelial cells resulted in the modulation of several miRNAs, with down-regulation of miR-375 in both epithelial cell types. In addition, miR-375 was down-regulated in esophageal biopsies from EoE patients. Esophageal miR-375 levels correlated with esophageal eosinophil levels and esophageal mast cell specific gene CPA3. Modulation of miR-375 level was sufficient to regulate IL-13 mediated epithelial gene expression, especially in the immunoinflammatory pathways. Focusing on the potential utility of miRNAs as biomarkers for allergic disease, we identified up-regulation of multiple miRNAs, most notably miR-146a, miR-146b and miR-223 in the plasma samples from EoE patients that could serve as both non-invasive diagnostic markers and potential epigenetic regulators of EoE. Collectively, these results demonstrate a key role of miRNAs in regulating germane pathways involved in allergic inflammation including polarization of the adaptive immune system, regulation of eosinophil production and differentiation and regulation of IL-13 induced responses. Advisors/Committee Members: Rothenberg, Marc.

▼ From flies to humans, Orthodenticle (Otd)-related homeodomain transcription factors are necessary for eye development. Vertebrates encode three Otd-related factors, Otx1, Otx2 and Crx, while Drosophila encodes a single factor, Otd. Mutations in Otx2 or Crx can lead to eye diseases by affecting morphogenesis or maintenance of photoreceptors. In the Drosophila eye, Otd has been found to play a conserved role and is likewise necessary for photoreceptor morphogenesis and subtype specification during terminal differentiation. This latter function occurs through cell-specific activation and repression of known target genes. However, the mechanism of how Otd regulates transcription remains unknown. To address this question, we performed an in vitro analysis of Otd to identify functional domains and found that Otd contains two independent activation domains, one at the N and C-terminus, and a potential repression domain centrally located in the protein. These results suggest that multiple domains of Otd contribute to regulating gene expression of different targets through different mechanisms. We also tested whether Otd plays a post-developmental role in photoreceptor maintenance, like Crx and Otx2 in vertebrates. Our preliminary results suggested that Otd may be important in the adult eye. We next investigated if the domains identified from the in vitro study were necessary in vivo for photoreceptor maintenance. Our results show that morphogenesis and maintenance of photoreceptors require different domains, and that the C-terminus is likely important for photoreceptor integrity. This system can further be used to study photoreceptor degeneration to better understand the mechanisms of eye disease. Advisors/Committee Members: Cook, Tiffany.

▼ During the development of the cardiovascular system, one of the key morphogenic processes is the development of the valves. Cardiac valve defects are one of the most common congenital cardiac malformations and have a high prevalence in adult onset valve disease. Treatment for cardiac valve malformations or valve disease is limited to surgical replacement, suggesting a need for improved understanding of the mechanisms involved in valve development and disease. Developing heart valves consist of an extracellular matrix (ECM) secreted by valve interstitial cells and surrounded by a layer of endocardial endothelial cells. During remodeling the valves elongate and the ECM is organized into three distinct layers. The atrialis/ventricularis layer contains elastin, the spongiosa layer is comprised of a mixture of proteoglycans, and the fibrosa layer consists of different types of densely packed collagen fibers. Diseased valves have an improper distribution and organization of these matrix proteins which produces dysfunctional valves. Matrix metalloproteinase-13 (MMP-13) is an enzyme found in the heart during development that is responsible for cleaving collagen fibers. It has also been shown to be upregulated in diseased heart valves along with other MMPs. Because collagen fibers provide strength and rigidity to the mature cardiac valve, it was hypothesized that increased production of MMP-13 would lead to disorganized ECM patterning and impaired valve function due to excessive degradation of collagen in valves, thus generating a murine model of degenerative valve disease. To test this hypothesis, the MMP-13 coding sequence was inserted into the CAG-CAT vector for conditional overexpression in mice. The CAG-CAT-MMP13 vector will be inserted into the mouse genome via random insertion, and the resulting mice will be bred with Tie2Cre or Col2a1Cre mice to produce overexpression of MMP-13. The effect of overexpressing MMP-13 will be assessed and compared with Cre-negative mice. It is expected that overexpression of MMP-13 will result in decreased collagen deposition and that ECM remodeling will be disrupted causing compromised valve function. Additionally, if double transgenic mice survive to late adulthood, development of valve disease will be assessed. It is expected that fibrillar collagen degradation by MMP-13 will cause valve dysfunction in mice which will be predisposed toward the development of cardiac valve disease. Analysis of these embryos and mice will provide new information on the remodeling stage of heart valve development and possibly on the progression of valve disease. Generation of animals that mirror development of cardiac malformations that progress to valve disease in humans is useful for future studies of valve repair and pathogenesis as well as the study of new treatments. Advisors/Committee Members: Yutzey, Katherine.

▼ Myeloid cells are a feature of most tissues. Recently, it has become clear that myeloid cells do much more than simply protect the host from foreign invaders. In the work presented here, myeloid cells (including macrophages and microglia) are shown to regulate blood vessel patterning, a function quite distinct from their role in innate immunity. Specifically, retinal myeloid cells (RMCs) are shown to produce non-canonical Wnt ligands as a means to suppress blood vessel branching in the retina. In response to these non-canonical Wnt ligands, RMCs secrete Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). This signaling cascade is not limited to the retina. In wound assays of repair and regeneration, myeloid cells are shown to regulate blood vessel patterning in a similar way. These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching in development and disease. This work, coupled with the emerging work of other laboratories, lays the groundwork for a new way of thinking about the immune system. Advisors/Committee Members: Lang, Richard.