Rodney Stewart, Ph.D. - US grants

The goal oflhis application is to use the zebrafish as a |nodel to dissect the molecular connponents of Foxd3-mediated neural crest (NC) cell survival and migration end to determine if these pathways promote metastasis in NG-derived tumors in vivo. The zebrafish with its close synteny to the human genome and its conserved molecular pathways regulating the development of tissues and organs, Offers a powerful tool with which to conduct such research. We previoulsy identified and characterized a zebrafish foxd3 mutant line that has specific NC ceil survival and migration defects. We also identified the Snai1b transcription factor-as a critical mediator of Foxd3 function. The underlying hypothesis of this applicatipn is that knowledge of the Foxd3 pathway wiII provide an understanding environments, requirement for the metaststasis of NC-derived tumors. In Aim 1, genetic epistasis and biochemistry will determine if Foxd3 directly activates snai1b expression. This aim will also will also determine whether Snai 1b inhibits NC apoptosis by repressing the expression of one or more BH3-only genes, and promotes NC migration by repressing cadherin-6 expression . In Aim 2, a list of genes identified in a microarray screen during the bK99 phase will abe analyzed for thier NC expression pattern and knockdown phenotypes, as well as their ability to rescue NC defects in foxd3 mutant and snai 1b MO-injected embryos. In Aim 3, foxd3 and snai1b will be expressed in developing NC cells to examine their potential roles in promoting metastasis in established zebrafish NC tumors, including a new model identified during the K99 phase. Rodney Stewart will continue to receive advanced training in genetics, neurobiology and oncology throughout his career. This research proposal is designed to support the candidate as an independent investigator during the first 3 years of a faculty appointment in the Department of Oncology Sciences at the Huntsman Cancer Institute at the University of Utah. Huntsrnan Cancer Institute at the yniyersity of-Utahnd

PROJECT SUMMARY ? HUNTSMAN CANCER INSTITUTE, RODNEY STEWART Craniofacial Disorders (CD) account for approximately one-third of all birth defects in children and are a primary cause of infant mortality. A major barrier to improved CD diagnosis and treatment is an incomplete understanding of the genetic and biophysical mechanisms that shape different craniofacial elements during embryogenesis. The highly orchestrated collective cell movements of the cranial neural crest (NC) lay the foundation for construction of cartilage and bones in the head and face, and when impaired causes CD, including misplaced, reduced or missing craniofacial elements. The overall objective of the current project is to determine if biophysical forces are required to target collective cranial NC cell populations, also called NC streams, to precise locations in the head to drive craniofacial morphogenesis. Defining how craniofacial morphogenesis is orchestrated at the biophysical level is likely to reveal potential causes of human craniofacial disorders and identify new treatment strategies. The central hypothesis is that compression forces originating from the neural tube act on a sheet-like NC population to guide the second NC stream. Guided by published and preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) generate a NC-specific tension sensor to measure forces within cranial NC streams and 2) determine if compression forces control migration of the second NC stream. The approach is innovative because it generates new tools to measure biophysical forces in vivo by taking advantage of the optical clarity and powerful imaging attributes of the zebrafish system using live embryos. The proposed research is significant because it is expected to advance and expand our understanding of how biophysical forces guide collective cell migration in many developmental and disease contexts, including craniofacial disorders, tissue regeneration and cancer invasion. In addition, the tools and data generated in this proposal will allow us to interrogate the genetic and cellular mechanisms controlling chemokine-dependent and biophysics-dependent targeting of cranial NC cells in a future R01- funded proposal, which is ultimately needed to reach our long-term goal of identifying treatments that can prevent or restore diseased craniofacial tissue.

PROJECT SUMMARY ? HUNTSMAN CANCER INSTITUTE, RODNEY STEWART Embryonic morphogenesis mechanisms are frequently activated in human cancers, particularly childhood brain tumors. In particular, genes that regulate neural crest (NC) epithelial-to-mesenchymal transition (EMT) and cell migration, such as the Snail family of transcription factors, are often re-activated in tumor cells to promote malignant progression by changing the available repertoire of cell adhesion, cytoskeletal, apoptotic and signaling molecules. This leads to enhanced tumor-cell dissemination, self-renewal and chemo-resistance, a devastating combination that promotes treatment failure and cancer-related mortality. The long-term goal of this research is to define the essential, conserved and rate-limiting effectors of embryonic EMT programs that drive cancer progression in order to identify new targets and therapeutics that can be used alone or in conjunction with current therapeutics to eliminate tumor cells. The overall objective of the current project, which represents the next logical step toward our long-term goal, is to 1) use the powerful genetic attributes of the zebrafish system to identify new mechanisms driving NC EMT and cell migration and 2) determine if inhibiting one or more EMT effectors in a model of pediatric brain tumors prevents tumor invasion and/or dissemination. The central hypothesis is that a subset of cell adhesion, cytoskeleton and/or signaling molecules are essential for executing NC EMT and that these molecules represent promising targets to inhibit EMT-induced brain tumor invasion. Guided by published and preliminary data, this hypothesis will be tested by pursuing three specific aims in which we will: 1) determine if the highly conserved Foxd3 transcription factor directly controls the expression of Snail genes during EMT, 2) identify essential effectors of Foxd3/Snail-dependent EMT during NC development and 3) determine if one or more NC effectors are required for brain tumor invasion in vivo. The proposal is innovative because it employs new genetic and imaging technologies to dynamically measure and manipulate the impact of developmental EMT programs during brain tumor invasion in whole animals at single-cell resolution for the first time, allowing the rapid identification of new targets and therapeutics for brain cancers. The successful completion of the proposed research will have a significant impact because it is expected to vertically advance and expand our understanding of how to manipulate developmental EMT programs in a number of disease settings, including NC-derived birth defects, fibrosis and cancer invasion, thus allowing for the strategic design of effective treatments for these diseases.