Mechanistic and target study of anthraquinone modifier KA-4c triggering endoplasmic reticulum stress to inhibit triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is associated with poor prognosis and high rates of metastasis and recurrence owing to lack of targeted therapies. Therefore, identifying effective targets for TNBC therapy remains an important clinical challenge. In this study, we examined the potential anticancer mechanisms and targets of the anthraquinone modifier KA-4c. Hematoxylin/eosin (HE), ER-Tracker™ Red, and Mito-Tracker™ Green staining followed by transmission electron microscopy (TEM) were performed to study the effect of KA-4c on TNBC cell morphology. KA-4c-induced apoptosis was detected using flow cytometry, and apoptosis-related proteins were analyzed using western blotting. Drug affinity-responsive target stability (DARTS), liquid chromatography tandem mass spectrometry (LC-MS/MS), bioinformatics, cell thermal shift analysis (CETSA), and RNA interference were used to identify the target protein of KA-4c. The results revealed increased cytoplasmic vacuolation from the endoplasmic reticulum and mitochondria in MDA-MB231 and MDA-MB468 cells treated with KA-4c. Furthermore, KA-4c enhanced MDA-MB231 and MDA-MB468 cell apoptosis by upregulating CHOP and caspase7 expression, and inducing PARP cleavage. DARTS results revealed that KA-4c activates the ER protein-processing signaling pathway by binding to ATF6 and rendering it resistant to protease hydrolysis. CETSA results demonstrated that KA-4c enhances ATF6 protein expression in a concentration-dependent manner. The results of RNA interference indicated that silencing ATF6 could effectively inhibit the upregulation of CHOP. In conclusion, KA-4c activates the ER protein-processing signaling pathway by targeting ATF6, damaging mitochondria, and inducing TNBC cell apoptosis. Thus, ATF6 represents a potential target of KA-4c, and a therapeutic target for TNBC.