Chemotherapy-induced Exosomal MiRNA Promote Breast Cancer Stemness

ObjectiveBreast cancer is the most common cancer in women,as well as a serious threat to women's health.Neoadjuvant chemotherapy is principally used in cancer treatment,but unfortunately fail to eradicate disease.One of the vital reasons may due to the induction of cancer stem cells(CSC)in chemo-drugs treatment,which resulting in tumor recurrence and poor prognosis.However,the influence and mechanisms remain unclear.Exosomes are a subset of extracellular vesicles with a typical diameter of 30-150nm,which are released by all cell types,including breast cancer cells.Serving as vehicles to transfer a variety of cellular materials including RNA,DNA,proteins,and lipids between adjacent or distant cells,exosomes have been recognized for their unique role in intercellular communication.Many reported functions of exosomes in the regulation of target cells are mediated by microRNAs(miRNAs).Cancer-derived exosomal miRNAs play vital roles in regulating self-renew and differentiation of CSCs.Here we showed that following treatment with a sub-lethal dose of chemotherapeutic agents,breast cancer cells secreted exosomes with the capacity to stimulate a CSC phenotype,rendering cancer cells resistance to the cytotoxic drugs.Methods1.Exosomes are derived from breast cancer cells MDA-MB-231,MCF-7 and BT474,as well as normal human breast cells MCF 10A in the presence of docetaxel,doxorubicin or PBS,and then treated recipient cells respectively.Sphere formation assay and ALDEFLUOR assay are conducted to analysis the CSC properities.The expression of stemness-related genes including NOTCH1,SOX9,NANOG and OCT4 in recipient cells was tested by RT-PCR and westernblot.2.Small RNA Seq was used to exam exosomal miRNA levels in chemotherapy-or PBS-induced MDA-MB-231 cells.The up-regulations of miR-9-5p,miR-203a-3p and miR-195-5p were further proved by RT-PCR in MDA-MB-231,MCF-7,BT474 and MCF 10A cells.The expression of their promising gene target ONECUT2 is analyzed by westernblot and RT-PCR.3.Sphere formation and ALDEFLUOR Assay were used to analysis CSC traits.MRNA and protein levels of ONECUT2 and stemness-associated genes were tested following treatment of specific miRNAs over-expression,ONECUT2 down-or up-regulation.4.Luciferase reporter assay were conducted to exam if ONECUT2 is the common target of miR-9-5p,miR-203a-3p and miR-195-5p.5.NSG mice were injected MDA-MB-231,MDA-MB-231/Rab27aKD,or MDA-MB-231/OC-2 cells.When the tumor volume reached 300mm~3,mice were treated weekly with DTX(15mg/kg)for 3 weeks.Serum-derived miRNAs and tumor tissue-derived mRNA pre-and post-chemotherapy were analysis by RT-PCR.The expression of ONECUT2 and stemness-related genes in tumor tissue was also tested by westernblot.IHC was conducted to analyze Ki-67,ONECUT2 and SOX9expressions in tumor tissues pre-and post-DTX treatment.6.MTS assay were conducted to test the chemotherapy resistance of MDA-MB-231cells in the situation of ONECUT2 down-regulation or three miRNAs overexpression.Results1.Cancer-derived exosomes and their cargo miRNAs secreted by chemotherapy-treated breast cancer cells induce a CSC phenotype and expression of stemness-related genes including NOTCH1,SOX9,NANOG and OCT4.2.Exosomal miR-9-5p,miR-203a-3p and miR-195-5p were elevated in chemotherapy-induced MDA-MB-231,MCF-7 and BT474.ONECUT2 terms to be the target gene of aforementioned miRNAs.Inhibition of these miRNAs or restoration of ONECUT2 expression abolished the CSC-stimulating effect of exosomes derived from chemotherapy-treated cancer cells.3.ONECUT2 inhibition or three specific miRNAs overexpression can induce CSC traits in MDA-MB-231,MCF-7 and BT474 cells.4.ONECUT2 is the common target of miR-9-5p,miR-203a-3p and miR-195-5p.5.In mice bearing xenograft mammary tumors,docetaxel treatment caused elevations of miR-9-5p,miR-195-5p,and miR-203a-3p in circulating exosomes as well as decreased ONECUT2 expression and increased levels of stemness-associated genes in MDA-MB-231 mice.These effects following chemotherapy were absent when the tumors were deficient in exosomes secretion or in ONECUT2overexpression.6.ONECUT2 inhibition or three specific miRNAs overexpression can induce chemotherapy resistance in breast cancer cells.In aforementioned situation,ABCB1,ABCC1,ABCG2 and ABCB5 showed significant up-regulations.Conclusions1.Chemotherapy induced breast cancer cells to secrete multiple exosomal miRNAs including miR-9-5p,miR-203a-3p and miR-195-5p,which simultaneously down-regulate transcription factor ONECUT2,leading to the induction of CSC traits including increased sphere formation efficacy and ALDH-positive breast cancer cells,as well as the upregulations of stemness-associated genes.2.Knockdown of ONECUT2 in breast cancer cells led to increased cancer stemness characteristic and drug resistance,which at least partially caused by the promotion of ABC transporters.