Cryptotanshinone Directly Targets MEK1 to Inhibit Migration and Invasion of Breast Cancer Cells Through Down‐Regulating ERK / EMT Axis

Cryptotanshinone (CPT) exerts inhibitory effects on breast cancers (BC). However, its effect and potential mechanism on breast cancer cell migration and invasion are not well understood. This study aims to investigate the potential mechanism of CPT on migration and invasion by network pharmacology and experiments. Wound healing and Transwell Matrigel invasion assays were performed to analyze the effects of CPT on migration and invasion in breast cancer cell lines MCF-7 and 4 T1. Western blot and immunofluorescence analyses were performed to verify the effect of CPT on epithelial-mesenchymal transition (EMT). Network pharmacology and experiments were used to explore the potential mechanism of CPT on EMT in breast cancer. Molecular docking, molecular dynamics (MD) simulation, cellular thermal shift assays (CETSA), drug affinity responsive target stability (DARTS), transfection, and mutagenesis were performed to confirm the direct binding effect of CPT on MEK1. Finally, BC nude mouse xenografts were established to explore the anti-metastasis effect of CPT in vivo. We found that CPT inhibited the EMT process to attenuate the migration and invasion of BC cells in a dose-dependent manner. Subsequently, network pharmacology, CETSA, DARTS, and enzyme activity assays confirmed that CPT directly binds MEK1 to inhibit its phosphorylation and kinase activity. Moreover, the molecular docking, MD, and mutagenesis results showed that CPT bound to MEK1 at Gly80 and Asp208. Moreover, MEK inhibitor (AZD-6244) intervention studies confirmed that CPT inhibits EMT by targeting the MEK pathway. Finally, the mouse tail vein injection model showed that CPT can effectively inhibit BC metastasis in vivo. Our results demonstrated that CPT modulates breast cancer migration and invasion via the MEK/ERK/EMT axis by directly targeting MEK1. Our study provides evidence suggesting that CPT may serve as a potential agent for suppressing breast cancer metastasis.