Essential Role Of UCH-L1-containing Exosomes In Breast Cancer With Chemotherapeutic Resistance

Chemoresistance has become a serious challenge in the treatment of breast cancer.Previous studies showed that cells can transfer proteins,including those responsible for drug resistance to adjacent cells via exosomes.In our study,we sought to analyze the role of UCH-L1-containing exosomes in the transfer of drug-resistance and to determine the diagnostic implications.Our study used adriamycin-sensitive human breast cancer cells(MCF7/WT)and adriamycin-resistant human breast cancer cells(MCF7/ADM)to investigate the mechanism of chemoresistance.CellTiter-Blue(CTB)Viability assay showed the MCF7/ADM cells displayed a 40-fold greater resistance to adriamycin than the parental wild-type cells line(MCF7/WT).We chose adriamycin to monitor the subcellular distribution of chemotherapeutic drugs due to its natural red fluorescence.Adriamycin was accumulated in the nucleus of the MCF7/WT cells.However,in MCF7/ADM cells,adriamycin was detected a small accumulation,the mostly remaining part of adriamycin located in cytoplasm.We used western blot analysis to determine the ubiquitin carboxyl terminal hydrolase-L1(UCH-L1),p-glycoprotein(P-gp),extracellular-signal regulated protein kinase1/2(ERK1/2)and phospho-extracellular-signal regulated protein kinase1/2(p-ERK1/2)protein expression levels in MCF7/ADM cells,MCF7/WT cells and UCH-L1-silencing MCF7/ADM cells(MCF7/ADM siRNA).The results showed that the UCH-L1,P-gp proteins expression levels and ERK1/2 phosphorylation levels were significantly higher in MCF7/ADM cells compared with MCF7/WT and MCF7/ADM siRNA cells.These results displayed that MCF7/ADM cells overexpressed UCH-L1 and P-gp compared to MCF7/WT cells.The switches of drug resistance via exosomes transfer were assessed by CTB Viability assay,flow cytometry(FCM)and immunostaining analysis.The exosomes were secreted from MCF7/ADM cells and carried UCH-L1 and P-gp proteins into the extracellular microenvironment then integrated into MCF7/WT in a time-dependent manner,transferring the chemoresistance phenotype.Following the exosome transfer,we assessed the chemoresistance in recipient MCF7/WT cells via CTB assays,and found a 7-fold higher resistance than control cells.To determine the function of UCH-L1 in the process of exosome transfer,we blocked the activity of UCH-L1 with the UCH-L1 specific inhibitor LDN-57444.We pretreated the exosomes from MCF7/ADM cell culture medium with LDN-57444,and then incubated them with MCF7/WT cells.The CTB assays revealed no significant difference in the chemosensitivity of MCF7/WT cells compared with controls.Furthermore,we measured the expression of UCH-L1 and P-gp in MCF7/WT cells after incubation with exosomes.FCM analysis showed UCH-L1 and P-gp immunofluorescence was increased in MCF7/WT cells after incubation with exosomes from MCF7/ADM cells,and we found that the expression P-gp was decreased in MCF7/WT cells when the exosomes were pretreated with LDN-57444,compared to those without LDN-57444.Since it has been shown that UCH-L1 activated the MAPK/ERK signaling pathway to enhance the expression of P-gp in MCF7/ADM cells,we further investigated whether this mechanism existed in the processes of transfer chemoresistance.Consistent with previous results,the expression of p-ERK was increased in MCF7/WT cells after incubation with exosomes that were secreted from MCF7/ADM cells.These results strongly implied that UCH-L1-containing exosomes transferred chemoresistance to MCF7/WT cells,not only carrying UCH-L1 to recipient cells but also enhanced the expression of P-gp via the MAPK/ERK signaling pathway.The exosomes mediated crosstalk resulted in increased expression of UCH-L1 and P-gp in MCF7/WT cells,which conferred higher resistance to Adriamycin.Immunohistochemistry was performed on 93 breast cancer samples to assess the associations of UCH-L1 levels with immunofluorescence value of UCH-L1 in circulating exosomes.Notably,in blood samples from patients with breast cancer,the level of exosomes carrying UCH-L1 before chemotherapy was significantly negatively correlated with chemotherapy outcome.Our study demonstrated that UCH-L1-containing exosomes can transfer chemoresistance to recipient cells and these exosomes may be useful as non-invasive diagnostic biomarkers for detection of chemoresitance in breast cancer patients,achieving more effective and individualized chemotherapy.