Atractylenolide I Blocks Lung Metastasis of Triple‐Negative Breast Cancer by Inhibiting SPP1 ‐Mediated Extracellular Matrix Signaling Pathway

Atractylodes macrocephala Koidz. has long been used for invigorating Qi and traditionally prescribed for cancer treatment by Chinese medicine practitioners. Atractylenolide I (ATI) is a major active component in A. macrocephala and has inhibitory effects on various malignancies. Triple-negative breast cancer (TNBC)-related deaths are predominantly attributed to metastasis. However, the activity and mechanism of ATI on TNBC metastasis are unclear. Cell viability was detected by CCK-8 assay. Cell migration was determined by wound healing and transwell assays. The in vivo experiment was performed in 4 T1 spontaneous metastasis breast cancer nude mice. The mechanism was investigated by RNA-seq analysis, and further verified by bioinformatics, qPCR, western blot, and siRNA transfection analysis. ATI inhibited the growth of 4TI cells. Additionally, ATI repressed the wound healing, invasion, and migration activities of 4TI and MDA-MB-231 cells at a lower concentration. In vivo experiment revealed that while ATI (50 mg/kg) did not significantly inhibit tumor growth but markedly suppressed lung metastasis progression in a 4 T1 spontaneous metastasis mouse model. RNA-seq analysis of lung tissues showed that the extracellular matrix (ECM) signaling pathway was significantly suppressed by ATI. In addition, ATI inhibited the mRNA and protein levels of ECM-related members (SPP1, MMP9, and COL1A1) in vitro and in vivo. Bioinformatics analysis revealed that the overexpression of SPP1 correlated with the poor prognosis of breast cancer patients. Furthermore, ATI inhibited ECM activator pyrintegrin-induced wound healing. siRNA knockdown of SPP1 abolished ATI's inhibition on wound healing, cell migration, and expression of ECM-related genes/proteins in 4 T1 cells. Molecular docking showed that ATI interacted with Trp-106, Pro-44, and Leu-46 residues in SPP1 (-7.73 kcal/mol). Our findings suggest that ATI could suppress the lung metastasis of TNBC by inhibiting the SPP1-mediated ECM signaling pathway.