Flubendazole elicits anti-cancer effects via targeting EVA1A-modulated autophagy and apoptosis in Triple-negative Breast Cancer.

Abstract

Background: Triple-negative breast cancer (TNBC) is one of the most prevalent neoplastic diseases worldwide, but efficacious treatments for this pathological condition are still challenging. The lack of an effective targeted therapy also leads to a poor prognosis for patients affected by TNBC. In the present study, we repurposed the distinctive inhibitory effects of flubendazole, a traditional anthelmintic drug, towards the putative modulation of proliferation and migration of TNBC in vitro and in vivo.Methods: According to a series of experimental approaches, including immunofluorescence (IF), immunoblotting (IB), siRNA and GFP-mRFP-LC3 plasmid transfection, respectively, we have found that flubendazole is capable of inducing autophagic cell death and apoptosis, thus exerting some anti-proliferative and anti-migration activity in TNBC cells. The therapeutic effects of flubendazole were evaluated by xenograft mouse models, followed by immunohistochemistry (IHC), IF and IB. Changes in the gene expression profiles of flubendazole-treated TNBC cells were analyzed by RNA sequencing (RNA-seq) and validated by IB. The potential binding mode of flubendazole and EVA1A was predicted by molecular docking and demonstrated by site-directed mutagenesis.Results: We have presently found that flubendazole exhibits a considerable anti-proliferative activity in vitro and in vivo. Mechanistically, the induction of autophagic cell death appears to be pivotal for flubendazole-mediated growth inhibition of TNBC cells, whereas blocking autophagy was able to improve the survival rate and migration ability of flubendazole-treated TNBC cells. Specifically, RNA-seq analysis showed that flubendazole treatment could promote the up-regulation of EVA1A. Flubendazole may regulate autophagy and apoptosis by targeting EVA1A, thus affecting the mechanisms of TNBC proliferation and migration. Furthermore, Thr113 may be the key amino acid residues for the binding of flubendazole to EVA1A.Conclusion: Our results provide novel insights towards the putative anti-cancer efficacy of flubendazole. Furthermore, here we show that flubendazole could serve as a potential therapeutic drug in TNBC. Altogether, this study highlights the possibility of this repurposed autophagic inducer for future cancer treatments.