Understanding the mechanisms by which environmental arsenic elevates the risk of urothelial bladder cancer

Arsenic (As) in drinking water is associated with increased risk of multiple malignancies, particularly urothelial bladder cancer (UBC), yet the underlying mechanisms remain poorly understood. We previously demonstrated that zebrafish possess a contractile urinary bladder with a specialized urothelium closely resembling the human bladder structurally and functionally. We and others have further shown that zebrafish As metabolism parallels that of humans and that zebrafish express the key As metabolism genes AS3MT and MTHFR. AS3MT is essential for methylating inorganic As into less toxic metabolites, while MTHFR is required for generating the universal methyl donor SAM; hypoactive SNPs in both genes are associated with increased UBC risk. Using both adult zebrafish and human cell lines, we aim to investigate how As impacts the bladder urothelium and elevates UBC risk. In a 28-day study, adult zebrafish exposed to 500 ppb As (III) exhibited histological evidence of As-induced hyperplasia and urothelial thickening. Bulk RNA-seq of As-exposed zebrafish bladders revealed upregulation of cancer-related pathways including MYC, mTOR, and unfolded protein response. Single cell RNA-seq further identified female-specific urothelial and non-urothelial clusters and revealed distinct clusters altered by As that differed between sexes, suggesting a potential link to the known sex bias in UBC. In both female and male urothelial clusters, As induced a shift of cells from a UPK+ phenotype to a less-differentiated UPK- phenotype. Loss of both luminal and basal markers including UPK3B, KRT5, and KRT15 was observed, while lineage-regulating factors like FOXA1 are retained. Induction of the AP-1 transcriptional complex, implicated in promoting tumor growth and invasion, was also observed. To validate, we employed the spontaneously immortalized human urothelial cell line HBLAK. While HBLAK cells are homogeneously basal, they can be induced with EGFR inhibition and PPARgamma activation to express luminal markers, providing a platform to study urothelial differentiation. Upon induction, As-exposed HBLAK cells exhibited a dose-dependent decrease in both basal (KRT14) and luminal (UPK2) markers while retaining FOXA1 expression, accompanied by upregulated AP-1 components, consistent with zebrafish in vivo findings. As also induced global loss of histone and DNA methylation, with reduced SAM levels. We will employ newly generated AS3MT and MTHFR knockout zebrafish lines and human urothelial cell lines to understand As-induced epigenetic dysregulation and its link to disrupted differentiation. Our findings establish zebrafish as a powerful model for studying As-induced changes in urothelial differentiation and suggest that As induces urothelial cell dedifferentiation in both zebrafish and human models, which may lead to the early emergence of tumor-initiating cells during tumorigenesis.