Astragaloside IV inhibits the growth of obesity-associated triple-negative breast cancer by activating FOXA1 transcription factor to regulate GAL3ST1-GalCer signaling and remodel sphingolipid metabolism

Background: Obesity is linked to a heightened risk of developing triple-negative breast Cancer (TNBC). Obesity-associated triple-negative breast Cancer (OA-TNBC) presents limited therapeutic options and is characterized by a poor prognosis. Although Astragaloside IV (ASIV) exhibits anti-tumor properties across different cancers, its effect and mechanisms in OA-TNBC remain unclear.

Purpose: The aim of this research was to explore the efficacy and underlying molecular mechanisms of ASIV in treating OA-TNBC.

Methods: The effects of ASIV on OA-TNBC was evaluated through analysis of cell cytotoxicity and colony formation assays. Oil Red O staining and immunofluorescence were employed to detect the effect of ASIV on lipid droplet formation. The xenograft experiment was carried out to assess the effect of ASIV on OA-TNBC. Lipidomics analysis was conducted to investigate the role of ASIV in remodeling tumor lipid metabolism. Mechanistic experiments, including western blotting, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), mRNA stability, protein stability, enzyme-linked immunosorbent assay (ELISA), plasmid transfection, dual-luciferase reporter assay, DNA pull down assay, and Chromatin Immunoprecipitation (ChIP) assay were employed to uncover the regulatory mechanisms of ASIV on OA-TNBC.

Results: In vitro experiments demonstrated that ASIV significantly inhibited the viability and proliferation of OA-TNBC cells, accompanied by decreased lipid droplet formation and lipid content. Furthermore, ASIV inhibited tumor growth without causing obvious toxicity to the liver, kidneys, and spleen. Lipidomics analysis of cells and tumor tissues revealed that sphingomyelin was the most significantly altered metabolite type. ASIV significantly regulated Galactosylceramide (GalCer) levels to remodel tumor sphingolipid metabolism. ASIV upregulated the expression of GalCer-metabolizing Enzymes galactose-3-O-sulfotransferase 1 (GAL3ST1) at both the mRNA and protein levels. Knockdown of GAL3ST1 attenuated the effects of ASIV on cell viability and proliferation, and the similar results were observed following exogenous supplementation of GalCer. ASIV had no significant effect on the stability of either GAL3ST1 mRNA or protein. The results of the dual-luciferase reporter gene assay demonstrated that ASIV significantly increased the activity of GAL3ST1 promoter. Moreover, GAL3ST1 is transcriptionally activated by transcription factor forkhead box A1 (FOXA1) that binds directly to the promoter region of the GAL3ST1 gene.

Conclusion: Our findings confirmed that ASIV could remodel tumor sphingolipid metabolism via activating the FOXA1 transcription factor to regulate GAL3ST1-GalCer signaling, thus displaying a promising therapeutic effect of ASIV in OA-TNBC.

Astragaloside IV; FOXA1; GAL3ST1; Obesity-associated triple-negative breast cancer; Sphingolipid metabolism.