Mechanism Of Repurposing Drug Flubendazole Elicits Proliferation And Metastasis Inhibition Via Targeting EVA1A To Regulate Autophagy In Breast Cancer

BackgroundBreast cancer is one of the most common malignancies worldwide and is also considered the leading cause of cancer-related death among women.Clinical treatments mainly include surgery,chemotherapy,radiotherapy and endocrine therapy.The lack of effective targeted therapy has led to severe adverse prognostic reactions,including recurrence and metastasis,making its clinical treatment face enormous challenges.Therefore,it is imperative to develop new treatment methods and drugs to improve the prognosis of breast cancer patients.Autophagy is an automated process in which aggregated proteins and aged（or malfunctioning）organelles are separated by double-membrane autophagosomes and then degraded in autolysosomes.In the context of cancer progression,it is worth noting that autophagy is considered a“double-edged sword.”Anti-cancer treatment can usually induce autophagy to prolong the cancer cell survival by removing damaged organelles and thus recovering nutrients after anti-cancer treatment.In addition,autophagy also regulates tumor proliferation and metastasis through self-degradation.Recently,a series of small molecule compounds targeting autophagy have shown distinct effects in cancer.Drug repurposing is a strategy that seeks new mechanisms or new uses for approved drugs.Compared with the development of new drugs,drug repurposing can shorten the timelines and costs of drug development.Significantly,drug repurposing can reduce the risk of drug development failure and become an attractive approach to improve cancer therapy.Flubendazole,a broad-spectrum anthelmintic drug,has been repurposed as a promising anti-cancer agent and potent inducer of autophagy.However,to our knowledge,minimal data has been generated regarding the induction of autophagy in breast cancer by flubendazole,and the intricate autophagy-related mechanisms underpinning the anti-cancer effect of flubendazole remain to be further defined.ObjectiveTo explore the anti-proliferation and anti-metastasis effects of flubendazole in breast cancer and clarify the mechanisms of autophagy and mitophagy,provide novel insights for the clinical therapy of breast cancer.Methods1.Anti-proliferation effects of flubendazole were determined by MTT assay,colony formation assay and LDH assay;2.Apoptosis induced by flubendazole were determined by Annexin-V/PI dual staining and TUNEL assay;3.Anti-migration effects of flubendazole were determined by Scratch and Transwell assays;4.Autophagy and autophagy flux induced by flubendazole were determined by electron microscope and GFP-m RFP-LC3 adenovirus transfection,respectively;5.The changes of mitochondrial membrane induced by flubendazole were determined by JC-1 staining and m PTP opening assays;6.The mitochondrial oxidative stress induced by flubendazole were determined by flow cytometry with Mito SOX^TM Red FM;7.The expression of related proteins induced by flubendazole were determined by immunoblotting and immunohistochemistry analysis;8.The fluorescence intensity and co-localization of related proteins induced by flubendazole were determined by immunofluorescence;9.Subcutaneous xenograft model and intravenous xenograft model to investigate the effects of flubendazole in vivo;10.The differentially expressed genes in breast cancer cells induced by flubendazole were detected by RNAseq analysis.Results1.Flubendazole induces autophagy-related cell death and elicits anti-proliferation and anti-metastasis effects in breast cancerFirstly,we evaluated the anti-cancer effect of flubendazole in various tumor cells and screened breast cancer as a candidate tumor for this study.Next,we demonstrated that flubendazole exhibits a considerable anti-proliferative and anti-metastasis activity in vitro and in vivo.Mechanistically,we found that flubendazole can induce apoptosis and autophagy in breast cancer cells.We also found that autophagy defects（si-ATG5）or autophagy inhibitors（3-MA）were able to improve the survival rate and migration ability of flubendazole-treated breast cancer cells according to a series of experimental approaches,indicating that flubendazole induces autophagy-related cell death and elicits anti-proliferation and anti-metastasis effects in breast cancer.2.Flubendazole induces autophagy and elicits anti-proliferation and anti-metastasis effects via targeting EVA1A in breast cancerRNA-seq analysis showed that flubendazole treatment could promote the up-regulation of EVA1A.Moreover,Flubendazole may regulate ATG5-dependent autophagy by targeting EVA1A,thus affecting the mechanisms of breast cancer proliferation and metastasis.3.Flubendazole regulates DRP1-mediated mitophagy to induce mitochondrial dysfunction in breast cancer cellsStudies have found that flubendazole destroys the mitochondrial membrane and induces mitochondrial dysfunction in breast cancer cells.Mechanistically,we observed that flubendazole increased DRP1 expression,which activated the PINK1/Parkin signaling pathway to induce mitophagy.In addition,inhibition of DRP1 expression will reduce flubendazole-induced mitophagy,and mitochondrial membrane damage and mitochondrial dysfunction are alleviated,indicating that flubendazole regulates DRP1-mediated mitophagy and induces mitochondrial dysfunction in breast cancer cells.4.Flubendazole induces DRP1-mediated mitophagy and elicits anti-proliferation and anti-metastasis effects via targeting EVA1A in breast cancer cellsIn this part,we found that the anti-proliferation and anti-metastasis effects of flubendazole were inhibited in sh DRP1 cell lines,indicating that DRP1-mediated mitophagy regulates the anti-cancer effects of flubendazole.In addition,we proved that flubendazole regulates mitophagy and induces mitochondrial dysfunction via targeting EVA1A in breast cancer cells.At the same time,the combination of gene knockout and overexpression experiments proved that DRP1-mediated mitophagy is one of the critical factors for the overexpression of EVA1A to play an anti-cancer effect,indicating that flubendazole induces mitophagy and elicits anti-proliferation and anti-metastasis effects via targeting EVA1A in breast cancer cells.Furthermore,Thr113 may be the critical amino acid residue for the binding of flubendazole to EVA1A.ConclusionsIn summary,this study reported for the first time that flubendazole elicits proliferation and metastasis inhibition via targeting EVA1A to regulate autophagy and mitophagy in breast cancer,which provides novel insights towards the putative anti-cancer efficacy of flubendazole.Furthermore,this study highlights the possibility of this repurposed autophagic inducer for future breast cancer treatments.