Targeting the SMAD3/CISD2 axis suppresses bladder cancer progression by promoting ferroptosis in mesenchymal-like bladder cancer cells

The poor prognosis of bladder Cancer (BCa) is primarily attributed to the acquisition of invasive and metastatic capabilities by tumor cells through epithelial-mesenchymal transition (EMT), yet the dynamic alterations in Ferroptosis during EMT and their regulatory mechanisms remain unelucidated. This study is the first to reveal the mechanism by which the SMAD3/CISD2 signaling axis regulates Ferroptosis in mesenchymal-like bladder Cancer cells. Clinical sample analysis demonstrated significantly reduced expression of E-cadherin and upregulation of N-Cadherin and Vimentin in muscle-invasive bladder Cancer tissues (MIBC), with EMT-related marker levels correlating with overall survival rates in BCa patients. In TGF-β1-induced mesenchymal-like bladder Cancer cells, ferroptosis-related genes (GPX4, SLC7A11) were markedly elevated, alongside increased lipid peroxides (LPO) and glutathione (GSH) levels. However, mesenchymal-like bladder Cancer cells exhibited heightened sensitivity to the Ferroptosis inducer Erastin, showing more pronounced suppression of proliferation, elevated ROS, higher LPO and MDA levels, and reduced GSH, confirming their enhanced susceptibility to Ferroptosis. RNA-seq revealed significant downregulation of SMAD3 in Ferroptosis inducer treated mesenchymal-like bladder Cancer cells. SMAD3 knockdown further exacerbated Ferroptosis markers (elevated Fe²⁺, ROS, LPO; decreased GSH), inhibited migration, invasion, and proliferation-phenotypes reversible by Ferroptosis inhibitors. Mechanistically, ChIP-seq combined with RNA-seq demonstrated that SMAD3 regulates CISD2 expression by binding its promoter region, with clinical specimens confirming a positive correlation between SMAD3 and CISD2 expression. Functional rescue experiments showed that CISD2 overexpression in Smad3-knockdown mesenchymal-like bladder Cancer cells reversed abnormal increases in Fe²⁺, ROS, LPO, and MDA while restoring GSH levels, indicating that SMAD3 modulates Ferroptosis through a CISD2-dependent pathway. In vivo experiments further demonstrated that targeting the SMAD3/CISD2 axis significantly suppressed xenograft tumor growth and activated Ferroptosis. In conclusion, this study elucidates a novel mechanism by which the SMAD3/CISD2 axis dynamically regulates Ferroptosis through redox homeostasis reprogramming during EMT, providing a potential therapeutic strategy for targeting the progression of MIBC.