Introduction: Arsenic is a naturally occurring metalloid classified as a group 1 carcinogen in humans. Chronic environmental exposure to inorganic arsenic has been frequently associated with different types of cancer, including squamous cell carcinoma of the skin and lung and bladder cancers. The bladder is one of the identified target tissues for arsenic toxicity as some of the toxic metabolites of arsenic excreted through urine have been detected in organs, particularly in the bladder. Hence, the study aims to understand how arsenic causes bladder carcinogenesis and dissect the molecular signaling responsible for transforming normal bladder epithelial cells. Methods: To develop an in vitro model of arsenic (250 nM: 33 mg/L))-induced malignant transformation, we chronically exposed healthy bladder epithelial cells (HU-hTERT1) for over 12 months to a median physiological concentration of arsenic and the transformed (AST) cells like that observed in BCa patients. Cell proliferation, qRT-PCR, and Western blot analyses were used to assess the carcinogenicity effect of arsenic in TRT-HU1 cells. In vivo, xenotransplanted mice models were used to confirm the tumorigenicity potential of the transformed bladder epithelial cells. Computational Data and statistical analysis were performed. Results: Our results demonstrate that arsenic levels in the urine of our bladder cancer patient's cohort are significantly higher (4–6 fold) than that observed in healthy controls. Arsenic-exposed cells began to form anchorage-dependent colonies and -independent spheroids after four months, and the number of colonies increased with exposure time. Our transcriptomic and RPPA analysis suggested stem cell activation may be responsible for arsenic-induced transformation in bladder epithelial cells. In addition, we found a robust induction of ALDH1A1 and CD44 transcript and protein expression correlating with stem cell enrichment in arsenic-mediated transformation. Silencing the stem cell activators (ALDH1A1 and CD44) abrogates arsenic-induced malignant transformation. Finally, the tumorgenicity potential of the transformed bladder epithelial cells was confirmed in xenografted mice models. Conclusions: Our resultssuggested that stem cell activators may significantly facilitate the arsenic-exposed cells' survival advantage, enabling the healthy epithelial cells to reprogram into a cancer stem cell phenotype, leading to malignant transformation. SOURCE OF Funding: N/A
