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.