20(R)-Panaxatriol enhances METTL3-mediated m6A modification of STUB1 to inhibit autophagy and exert antitumor effects in Triple-Negative Breast Cancer cells

• Published in: Phytomedicine (Stuttgart) 2024-07, Vol.130, p.155537, Article 155537
• Main Authors: Li, Yan, Luo, Bingjie, Lin, Xuan, Bai, Donghui, Li, Lingyu, Gao, Duan, Li, Xiaoyun, Zhong, Xianxun, Wei, Yaru, Yang, Li, Zhu, Xiaofeng, Han, Li, Tian, Huaqin, Zhang, Ronghua, Wang, Panpan
• Format: Article
• Language: English
• Subjects: 20(R)-panaxatriol; Autophagy; METTL3; N6-methyladenosine; STUB1; Triple negative breast cancer
• ISSN: 0944-7113; 1618-095X; 1618-095X
• DOI: 10.1016/j.phymed.2024.155537

Aberrant activation of autophagy in triple-negative breast cancer (TNBC) has led researchers to investigate potential therapeutic strategies targeting this process. The regulation of autophagy is significantly influenced by METTL3. Our previous research has shown that the Panax ginseng-derived compound, 20(R)-panaxatriol (PT), has potential as an anti-tumor agent. However, it remains unclear whether PT can modulate autophagy through METTL3 to exert its anti-tumor effects. Our objective is to investigate whether PT can regulate autophagy in TNBC cells and elucidate the molecular mechanisms. For in vitro experiments, we employed SUM-159-PT and MDA-MB-231 cells. While in vivo experiments involved BALB/c nude mice and NOD/SCID mice. In vitro, TNBC cells were treated with PT, and cell lines with varying expression levels of METTL3 were established. We assessed the impact on tumor cell activity and autophagy by analyzing autophagic flux, Western Blot (WB), and methylation levels. In vivo, subcutaneous transplantation models were established in BALB/c nude and NOD/SCID mice to observe the effect of PT on TNBC growth. HE staining and immunofluorescence were employed to analyze histopathological changes in tumor tissues. MeRIP-seq and dual-luciferase reporter gene assays were used to identify key downstream targets. Additionally, the silencing of STIP1 Homology And U-Box Containing Protein 1 (STUB1) explored PT's effects. The mechanism of PT's action on STUB1 via METTL3 was elucidated through mRNA stability assays, mRNA alternative splicing analysis, and nuclear-cytoplasmic mRNA separation. In both in vivo and in vitro experiments, it was discovered that PT significantly upregulates the expression of METTL3, leading to autophagy inhibition and therapeutic effects in TNBC. Simultaneously, through MeRIP-seq analysis and dual-luciferase reporter gene assays, we have demonstrated that PT modulates STUB1 via METTL3, influencing autophagy in TNBC cells. Furthermore, intriguingly, PT extends the half-life of STUB1 mRNA by enhancing its methylation modification, thereby enhancing its stability. In summary, our research reveals that PT increases STUB1 m6A modification through a METTL3-mediated mechanism in TNBC cells, inhibiting autophagy and further accentuating its anti-tumor properties. Our study provides novel mechanistic insights into TNBC pathogenesis and potential drug targets for TNBC. Our study has revealed that in TNBC cells, PT can suppress autophagy and exert it