Cellular and Molecular Mechanisms of Arsenic-Induced Urothelial Bladder Cancer - ABSTRACT Urothelial bladder cancer represents a significant global public health burden, accounting for some 200,000 deaths each year. Among the many environmental risk factors for bladder cancer, drinking water contaminated by inorganic arsenic (iAS) represents a common cause, especially among rural populations. This mechanism appears especially relevant here in the rural state of New Hampshire, which suffers from the single highest incidence of bladder cancer among the fifty states. Despite longstanding knowledge that iAs exposure represents a highly preventable cause of bladder cancer, the precise mechanism by which iAs increases bladder cancer risk remains uncertain, leading to an absence of post-exposure risk mitigation strategies. To address this issue, we now propose to develop an entirely novel zebrafish model of urinary bladder cancer, allowing us to leverage the many advantages of the zebrafish model organism including facile gene targeting and a long history of productive use in environmental toxicology research. Our group has recently demonstrated that adult zebrafish harbor a mammalian-like contractile urinary bladder, with single cell (sc) RNA-Seq identifying both basal and luminal urothelial cell types similar to those observed in mouse and human. Based on these findings, we now propose to test the following central hypotheses: First, that urothelial-specific oncogene activation combined with tp53, kdm6a and stag2 inactivation will induce zebrafish urothelial neoplasia; second, that arsenic exposure will alter the transcriptional landscape and cell composition of zebrafish bladder; and third, that a combination of oncogenic stimuli and arsenic exposure will accelerate these changes in both pre-malignant and neoplastic zebrafish urothelium. To test these hypotheses, the following Specific Aims will be pursued: 1) To examine the ability of urothelial-specific oncogene activation and tumor suppressor gene inactivation to induce neoplastic transformation in zebrafish urinary bladder; and 2) To determine the effects of iAs on transcriptional landscapes and cell composition in normal, pre-neoplastic and/or neoplastic zebrafish urinary bladder. These studies will be enabled by our broadly inter-disciplinary, multi-institutional research team comprised of experts in zebrafish cancer modeling (Leach), bladder cancer molecular genetics (Real) and arsenic toxicology and single cell transcriptional profiling (Goodale), allowing us to pursue a variety of highly innovative strategies. Together, the studies proposed in this R21 application will determine the effects of combined oncogene activation and tumor suppressor gene inactivation in zebrafish urothelium, potentially leading to a valuable new zebrafish model of urinary bladder cancer. In addition, these studies will provide the very first glimpse of how inorganic arsenic alters the transcriptional landscape of urothelial and non-urothelial cells in vivo and at single cell resolution, informing future strategies for the effective prevention of arsenic-induced bladder cancer.
