Isocucurbitacin B targets STAT3 to induce ferroptosis and promote anti-PD1 immunotherapy responses in breast cancer.

[OBJECTIVES] This study examines the pharmacological mechanisms underlying the antitumor activity of isocucurbitacin B (IB) and evaluates its potential clinical applications in triple-negative breast cancer (TNBC).

[METHODS] Comprehensive preclinical evaluations of IB were conducted utilizing a range of breast cancer cell lines and in vivo models, such as MMTV-PyMT mice and patient-derived organoids. Multi-omics integration - including bulk and spatial transcriptomics, untargeted metabolomics, and proteomics - unveiled the molecular foundation of IB's antitumor efficacy. The ferroptosis phenotype was validated through biochemical hallmarks, such as increased reactive oxygen species, ferrous ion levels, lipid peroxidation, and changes in mitochondrial morphology and ferroptotic protein expression. Seahorse metabolic flux analysis illustrated IB-induced disruptions in the citric acid cycle and oxidative phosphorylation (OXPHOS). Label-free chemoproteomic strategies, including drug affinity response target stability-mass spectrometry, were employed to identify IB's direct protein targets, with mechanistic insights confirmed via chromatin immunoprecipitation-quantitative polymerase chain reaction and dual-luciferase reporter assays to assess STAT3 transcriptional activity. To explore IB's immunotherapeutic potential, the 4T1 syngeneic murine model was used, and underlying molecular mechanisms were elucidated using flow cytometry and spatial transcriptomics.

[RESULTS] IB exhibited cell lineage-dependent anti-proliferative effects, showing significant potency against TNBC cells through mechanisms that include pyroptosis induction, G2/M phase cell cycle arrest, and the suppression of epithelial-mesenchymal transition. Its cytotoxicity was shown to be ferroptosis dependent, as demonstrated by rescue assays using ferroptosis inhibitors and its synergistic lethality with ferroptosis inducers. B-induced ferroptosis, as confirmed by various ferroptosis hallmarks, included elevated ferrous iron levels and lipid peroxidation. It also altered ferroptotic protein markers, specifically reducing the levels of SLC7A11 and GPX4 while increasing ACSL4. By directly targeting STAT3, IB bound to the SH2 domain, hindering its activation and subsequent nuclear translocation. Additionally, a novel regulatory axis was discovered where STAT3 transcriptionally activates SLC47A1, thereby contributing to ferroptosis resistance. Moreover, IB worked synergistically with anti-PD1 inhibitor by enhancing the infiltration of CD8+ T cells through the suppression of the STAT3/PD-L1/PD-1 signaling axis.

[CONCLUSION] IB shows considerable potential as a dual-functional agent in breast cancer treatment by targeting both ferroptosis induction and STAT3 inhibition. This approach connects ferroptosis resistance to immune evasion, highlighting promising translational prospects for combinatorial immunotherapy strategies.