Doxorubicin is a cornerstone chemotherapeutic agent. However, its role in ferroptosis and the underlying molecular mechanisms in endometrial cancer (EC) remain inadequately explored. We integrated transcriptomic profiling, machine learning, and protein–protein interaction network analyses to identify ferroptosis-related regulators in EC. Functional validation was performed using CCK-8 and EdU assays, transmission electron microscopy, biochemical assays of ferroptosis markers, and Western blotting. Mechanistic studies included RNA sequencing, molecular docking, cellular thermal shift assays, and surface plasmon resonance. In vivo effects were evaluated in nude mouse xenograft models. Bioinformatic analyses identified ferroptosis-related genes and highlighted a hub gene closely associated with drug sensitivity, with doxorubicin emerging as the agent most strongly linked to ferroptosis-related sensitivity. Doxorubicin markedly suppressed the proliferation of EC cells. This antiproliferative effect was partially reversed by ferrostatin-1. Morphological and biochemical analyses revealed features consistent with ferroptosis, including mitochondrial structural damage, increased lipid peroxidation, iron overload, and oxidative stress. Consistently, doxorubicin downregulated ferroptosis-associated proteins. Mechanistic studies demonstrated that doxorubicin binds directly to MKK6, thereby suppressing activation of the MKK6/P38/CEBPB signaling cascade. CEBPB positively regulates SLC7A11 expression and transactivates the SLC7A11 promoter in a motif-dependent reporter assay. CEBPB overexpression reverses both the anti-proliferative effects of doxorubicin and doxorubicin-induced ferroptosis in EC cells. In vivo, doxorubicin significantly reduced xenograft tumor growth while increasing ferroptosis-associated biochemical and molecular markers and inhibiting MKK6/P38 signaling. Our findings uncover ferroptosis as a previously unrecognized mechanism of doxorubicin action in EC, establishing the MKK6/P38/CEBPB axis as a potential therapeutic target and opening new avenues for treatment optimization.
Zhang et al. (Mon,) studied this question.