Aucubin inhibits the activity of lung cancer stem-like cells by targeting degradation of β-catenin.

Objectives: To investigate the antitumor effects of aucubin (AC) in non-small cell lung cancer (NSCLC) and uncover its plausible mechanism against lung cancer stem-like cells (LCSCs).

Methods: In vitro experiments included MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a reagent commonly used for cell viability assay) and colony formation assays to assess anti-proliferative effects on A549 and NCI-H1975 lung cancer cell lines, wound healing and Transwell invasion assays to evaluate inhibition of cell migration and invasion, tumorsphere-formation experiments to detect changes in NSCLC cell stemness, as well as Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses to measure the expression of LCSC markers (CD44, CD133, Oct4, and Nanog). In vivo experiments were conducted to observe the impact of AC on NSCLC metastasis and mouse survival rates. Further mechanistic studies involved transcriptomic gene set enrichment analysis, Western blot, qRT-PCR, molecular docking, and Surface Plasmon Resonance (SPR) methods to investigate how AC directly targets β-catenin and promotes its ubiquitin-mediated degradation.

Key findings: AC exerted significant anti-proliferative effects on A549 and NCI-H1975 cells, inhibited cancer cell migration and invasion, reduced the stemness of NSCLC cells, and markedly downregulated the expression of LCSC markers in vitro. In vivo, AC treatment significantly reduced NSCLC metastasis and improved mouse survival rates.Mechanistically, AC blocked the WNT (Wingless-related integration site, a family of secreted lipid-modified signaling glycoproteins that play crucial roles in embryonic development, tissue homeostasis, and regeneration) signaling pathway by downregulating β-catenin and c-Myc expression. It directly targeted β-catenin, promoting its degradation via the ubiquitin-proteasome pathway.

Conclusions: This study uncovers a novel anti-LCSC mechanism of AC, offers alternative strategies for NSCLC treatment, and provides innovative lead compounds for the development of drugs targeting lung cancer stem cells.