The 6-methyladenine erasers ALKBH5 and FTO influence chemotherapy efficiency in bladder cancer cell lines

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Abstract
Background: Bladder cancer (BLCA) is a prevalent and life-threatening condition that significantly impacts patients’ quality of life while imposing substantial financial costs on healthcare systems. Advancing our knowledge of the mechanisms underlying tumor development is crucial for improving treatment outcomes. Emerging studies emphasize the critical role of the RNA modification 6-methyladenine (m6A) and its associated proteins, methyltransferase-like 3 (METTL3), Vir-like m6A methyltransferase associated (VIRMA) (writers), Alkb homolog 5 (ALKBH5) and fat mass and obesity associated protein (FTO) (erasers), in maintaining m6A homeostasis. Dysregulation of these enzymes leads to aberrant m6A methylation, a hallmark of various cancers, including BLCA. Furthermore, m6A modifications influence cisplatin sensitivity, a key drug in muscle-invasive bladder cancer (MIBC) treatment. With this background, we investigated the combined effects of ALKBH5 and FTO knock-down in bladder tumor cell lines. Methods: We first investigated the expression of METTL3, VIRMA, ALKBH5 and FTO in BLCA tissues and human bladder tumor cell lines from urinary cancer cells by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Simultaneous knock-down of the expression of the erasers was then performed to explore their consequences in bladder cells. We then conducted cisplatin and mitomycin (MMC) treatment in knock-down cells to decipher the effect of their reduction. The cell viability was evaluated with cell counting kit-8 (CCK-8) assay after the two treatment regimes. Results: Lower expression of ALKBH5 and FTO was identified in BLCA tissue and bladder tumor cell lines. Notably, this trend was consistent across both low-grade and high-grade tissue samples. Furthermore, lower expression levels of ALKBH5 and FTO were observed in tumor cell lines derived from both men and women compared to the non-tumorigenic SV-HUC1 cell line. In contrast, both tissue and cell line data revealed an increased expression tendency of the m6A writers METTL3 and VIRMA. Additionally, knock-down of the two m6A erasers was found to enhance tolerance to cisplatin and MMC treatment, resulting in increased resistance to cell death. Conclusions: Our findings reveal that ALKBH5 and FTO are down-regulated in BLCA and their knock-down confers resistance to cisplatin and MMC in vitro. This suggests that m6A erasers play a critical role in modulating chemotherapy sensitivity, potentially serving as biomarkers or therapeutic targets for enhancing treatment efficacy in BLCA.

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