Abstract
Introduction
Urinary bladder carcinoma is the fifth most common cancer among men in the United States, with an estimated 64,730 new cases and 12,640 deaths in 2026. High reoccurrence rate and continual monitoring make it one of the most expensive malignancies to treat. These factors drive an urgent need for novel targeted therapies to combat bladder cancer. Inhibiting glutathione peroxidase 4 (GPX4) to induce ferroptosis—an iron-dependent, non-apoptotic cell death pathway—presents a novel therapeutic strategy. While expression of ZEB1, an epithelial-to-mesenchymal transition transcription factor, correlates with GPX4 inhibitor (GPX4i) sensitivity in several cancers, its role in bladder cancer remains uncharacterized. We hypothesized that ZEB1 expression drives sensitivity to GPX4i induced ferroptosis in bladder cancer by altering iron availability.
Methods
We used a panel of eight bladder cancer cell lines (T24, TCCSUP, SW780, RT4, 5637, HT1376, UMUC3, HT1197) to characterize sensitivity to GPX4i compounds RSL3 and ML210. Ferroptosis specificity was confirmed using the ferroptosis inhibitor liproxstatin-1. Protein expression of ZEB1, CDH1, GPX4, ferritin heavy chain (FtH), and ferritin light chain (FtL) were measured by western blot. Baseline labile iron pool (LiP) levels were quantified using FerroOrange fluorescent probe to determine if ZEB1 expression correlates with iron availability.
Results
RSL3 demonstrated high potency, yielding nanomolar IC50 values in six of eight cell lines. ML210 demonstrated moderate potency, with nanomolar IC50 values in four of eight cell lines. Liproxstatin-1 rescued cell viability in seven of eight cell lines, confirming GPX4-induced ferroptosis in those lines. Contrary to expectations, GPX4i sensitivity did not significantly correlate with ZEB1 expression or GPX4, FtH, and FtL protein levels. Baseline LiP levels varied widely across bladder cancer cell lines, with ZEB1 low lines exhibiting higher LiP levels than ZEB1 high lines.
Conclusion
We characterized sensitivity to GPX4i-induced ferroptosis in a panel of bladder cancer cell lines, identifying RSL3 as a potent compound and confirming that bladder cancer is susceptible to GPX4i. Unexpectedly, ZEB1 expression is not always associated with ferroptosis sensitivity. UMUC3 had the highest ZEB1 expression, but liproxstatin-1 did not rescue viability, indicating that these cells are not dying from ferroptosis. Conversely, HT1197 had low ZEB1 expression and the highest sensitivity to RSL3. Cell lines SW780, RT4, and 5637 also have low ZEB1 expression yet are sensitive to GPX4i. These contradictions suggest a ZEB1-independent mechanism of ferroptosis induction. ZEB1 also did not correlate with FtH or FtL protein expression, suggesting that ZEB1 does not affect iron storage capacity of bladder cancer cells. ZEB1 expression was anti-correlated with LiP, refuting our hypothesis. Higher LiP in ZEB1 low cell lines could explain GPX4i sensitivity and ferroptosis induction. It also suggests that ZEB1 could be suppressing LiP as a preventative measure against ferroptosis. These findings broaden the potential patient population for GPX4-targeted therapies to include ZEB1 low non-mesenchymal tumors and highlight baseline LiP as an important factor in ferroptosis susceptibility. Future work includes establishing a ZEB1 knockout cell model to study the role of ZEB1 in bladder cancer iron metabolism.