CITED1 nuclear localization and its role in Wilms' tumor pathogenesis

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DESCRIPTION (provided by applicant): This Pathway to Independence Award outlines an initial two-year mentored training program followed by a three-year independent phase to expedite the transition to scientific independence for Dr. Lovvorn. In the initial mentored phase, Dr. Lovvorn proposes to expand his scientific skills and knowledge in the fields of Cancer and Developmental Biology through a multidisciplinary program under the supervision of Dr. Mark de Caestecker, a highly productive principle investigator and respected mentor at Vanderbilt University. With the endorsement of his Department Chair, Dr. Lovvorn currently is practicing a work effort formula that affords 75% of his weekday time for research purposes. The research plan detailed herein will provide Dr. Lovvorn with rigorous training in basic biochemistry techniques to test the functional significance of and mechanisms regulating nucleocytoplasmic shuttling of the transcriptional co-factor CITED1 in Wilms' tumor. Under Dr. de Caestecker's guidance, Dr. Lovvorn has shown that expression of,CITED1, normally active in the developing but absent in the mature kidney, persists in both human and rat Wilms' tumors. He also has shown that the subcellular localization of CITED1 shifts from the cytoplasm in metanephric mesenchyme to the nucleus of malignant blastema, and that increasing levels of CITED1 expression in primary human Wilms' tumors correlate with advanced stages of disease. Dr. Lovvorn proposes two hypotheses from these observations: 1. CITED1 nuclear localization facilitates Wilms' tumor pathogenesis, and 2. Nucleocytoplasmic shuttling of CITED1 is dysregulated in Wilms' tumorigenesis. In his initial aim, Dr. Lovvorn will test the pathogenic significance of restricting CITED1 to the nucleus of Wilms1 tumor cells. He will generate two Wilms' tumor cell lines that misexpress either a predominantly cytosolic or nuclear fraction of CITED1 in the human Wilms' tumor cell line, WIT49, and will assess the effects of these manipulations on Wilms' tumor biology. In his second aim, Dr. Loworn, using mass spectrometry, will identify CITED1 binding partners that are specific to either subcellular compartment. These data will facilitate identification of CITED1 modifications and mechanisms regulating its nuclear retention in Wilms' tumor blastema. These studies will provide a framework not only to understand altered CITED1 nucleocytoplasmic shuttling in, and its contributions to, Wilms' tumor pathogenesis, but also the necessary training to pursue future independent investigation.