Doxycycline Inhibits The Cancer Stem Cell Phenotype And Epithelial-to-mesenchymal Transition In Breast Cancer

Backgrounds Breast cancer（BC）is the leading site of new cancer cases and second in cancer deaths among women.In 2016,it is estimated that among U.S.women there will be 246,660 new cases of invasive breast cancer,which will result in 40,450 breast cancer deaths.Despite effective early detection and anti-tumor therapies,approximately 10-15%of patients will experience tumor recurrence with distant metastatic disease,the leading causes of death in breast cancer patients.Experimental evidence suggest that breast tumors originate from breast cancer stem cells（BCSCs）,and that mitochondrial biogenesis is essential for the anchorage-independent clonal expansion and survival of CSCs,thus rendering mitochondria a significant target for novel treatment approaches.One of the recognized side effects of the FDA-approved drug,doxycycline is the inhibition of mitochondrial biogenesis.Objective Here we investigate the mechanism by which doxycycline exerts its inhibitory effects on the properties of breast cancer cells and BCSCs,such mammosphere forming efficiency,invasion,migration,apoptosis,the expression of stem cell markers and epithelial-to-mesenchymal transition（EMT）related markers of breast cancer cells.In addition,we explored whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells.Methods We detect the inhibitory effect of doxycycline in breast cancer cells and cancer stem cells by CCK-8 assays.And explore the effect of doxycycline on apoptosis,stem cell markers by flow cytometry,also detect its effect on the expression of stem cell factor Nanog,Oct4,Sox2 and EMT related markers by Real-time PCR and western-blot.We also detect its effect on invasion and migration by transwell assays.In addition,we investigate whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells by western-blot.Results 1)We find that doxycyline can inhibit the viability of breast cancer cells and BCSCs,IC₅₀values for MCF-7 and MDA-MB-468 of 11.39μM and 7.13μM respectively,the IC₅₀values for BCSC-enriched mammosphere cultures increased by more than 3-fold for both lines,37.5μM and 29.1μM for MCF7 and MDA-MB-468,respectively;2)Doxycycline also could inhibit the clonogenic ability of breast cancer cells,and induce apoptosis after exposure to doxycycline for 72 hours.Early apoptotic cells(Annexin-V^pos/PI^neg)in the doxycycline treated group were significantly increased compared to vehicle treated groups in both cell lines（MCF7:p=0.0054;MDA-MB-468:and p=0.0021）;3)In addition,treatment with a single IC₅₀dose of doxycycline for 72hours significantly decreased the CD44⁺CD24^–/lowcell population in both MCF7 and MDA-MB-468,compared to untreated cells（p<0.05）.For MCF7,CD44⁺CD24^–/lowcells in the control group and doxycycline-treated group are 6.8%,2.6%repectively.For MDA-MB-468,CD44⁺CD24^–/lowcells in the control group and doxycycline-treated group are 33.2%,12.7%respectively.Doxycycline also could decrease the mammosphere forming efficiency and expression of stem cell factors Oct4,Sox2,Nanog of breast cancer cells;4)Doxycycline also could inhibit the expression of epithelial-to-mesenchymal transition related factors,invasion and migration of triple negative breast cancer cells;5)Moreover,doxycycline could down-regulate the expression of the autophagy marker LC-3BI and LC-3BII,suggesting that inhibiting autophagy may be responsible in part for the observed effects on proliferation,EMT and stem cell markers.Conclusion The potent inhibition of EMT and cancer stem-like characteristics in breast cancer cells by doxycycline treatment suggests that this drug can be repurposed as an anti-cancer drug in the treatment of breast cancer patients in the clinic.