Identification of ADP-ribosylation factor 6 as the cellular target of withangulatin A against TNBC cells by ferroptosis

Abstract

Abstract Purpose Effective anticancer therapy can be achieved by regulating ADP-ribosylation factor 6 (ARF6) activation, but its biological roles are still challenging and few effective compounds against ARF6 have been identified. WA exerts the anti-proliferative effects on MDA-MB-231 cells. However, its molecular targets and unexplored mechanisms remain elusive. In the present study, our objective was to develop novel therapeutic strategies to improve therapy effectiveness. Methods Using experiment approaches, including CCK8, MDC assay, colony formation assay, immunoblotting and immunohistochemistry, we verified the therapeutic effects of Withangulatin A (WA). ARF6 was identified as the potential target of WA by human proteome microarray analysis. The binding of WA and ARF6 was validated by Microscale Thermophoresis assay, immunoprecipitation, immunofluorescence and ARF6 GTP pulldown assay. The binding mode was analyzed by molecular docking and site-directed mutagenesis. Results WA potently inhibited the activation of ARF6, which regulated the anticancer effect of WA in triple negative breast cancer (TNBC). Mechanistically, the ferroptosis mediated by WA was found through statistical analysis of genomics. Furthermore, WA can regulate ferroptosis by targeting ARF6, thus affecting the proliferation of TNBC cells. Particularly, Gln67 and His76 may be the key amino acid residues to influence the binding of WA to ARF6. Interestingly, the synergistic effects of WA and 3-MA sensitized TNBC cells to ferroptosis induction. Conclusion Our study identified the cellular target of WA and revealed the unrecognized function of ARF6, and it would provide a promising alternative therapy for triple negative breast cancer.