Exosomal MicroRNA-221/222Enhances Tamoxifen Resistance In Recipient ER-positive Breast Cancer Cells

Nowadays, breast cancer is the most common cancer among women with the rapidlyincreasing and much younger incidence worldwide. Because breast cancer can besymptomless in most clinical settings, it is always delayed at the diagnosis, leading toprogressive disease deterioration and ultimately life-threating events. Comprehensivetreatments including endocrine therapy, surgical intervention, radiotherapy and themolecular targeted therapy extended the overall survival. Endocrine therapy, also knownas hormone therapy, is an important part of the comprehensive treatments of breast cancerand it has been widely appreciated. With the advantages of long-term effects, feweradverse effects and the higher life quality, endocrine therapy can be used as a salvagetherapy after recurrence or metastasis.Tamoxifen, an antagonist of the estrogen receptor (ER), competitively inhibits theinteraction of estrogen with the ER, thus repressing ER activity. It has been showed thatTamoxifen can greatly prolong disease-free survival and induce remission in patients withERα positive metastatic disease. However, most ERα positive breast cancer patientspresent with primary or secondary tamoxifen resistance.To date, several reports have detailed mechanisms of resistance to tamoxifen in cancer cells. Various mechanisms have been proposed, including loss or mutations of the ER,cross-talk between ER and growth factor receptor pathways and high levels of so-calledanti-estrogen binding sites. However, the mechanisms of tamoxifen resistance remainunknown and are likely multifactorial. Recent results support that abnormalities inmicroRNAs play a role in tamoxifen resistance and indicate that microRNA-221/222issignificantly upregulated in MCF-7TamRcells.Secreted microRNAs were first identified in human serum by Adrian Harris in2008and they have also been detected in several biological fluids including breast milk, salivaand urine. Unlike microRNAs extracted from tissues or cells, secretory microRNAs areresistant to RNaseA digestion. It has been demonstrated that the majority of microRNAsdetectable in serum and saliva are concentrated in exosomes. Exosomes, smallmembranous vesicles (30-100nm), are mainly derived from the fusion of multivesicularbodies with the plasma membrane and are thought to be secreted by a wide range of cellsand serve an important purpose in intercellular communication. However, whether is itinvolved in tumor drug resistance? To study the contribution of microRNA-221/222totamoxifen resistance in recipient ER-positive breast cancer cells, we conduct severalexperiments and report the following findings.Methods and results:1. MCF-7TamRand MCF-7wtcells release exosomes with different characteristics（1）The different characteristics of MCF-7TamRand MCF-7wtexosomes were tested by electronmicroscopy analysis.(2) Quantitation of average total amount of exosomal protein andRNA per106cells were tested by nanodrop-2000c. Results: MCF-7wtexosomes appearedas clusters of vesicles approximately65nm in diameter, with each vesicle surrounded by adouble-layered membrane. However, MCF-7TamRexosomes were smaller than MCF-7wtexosomes. The amounts of protein and RNA isolated from the exosomes of106cells weredramatically different. MCF-7wtcells had an increased amount of exosomal RNA andproteins compared with MCF-7TamRcells.2. Secreted exosomes from MCF-7TamRCells transfer tamoxifen resistance（1） Exosomes labeled with PKH67were observed with confocal microscopy.(2) The effect ofMCF-7TamRexosomes on transmission of tamoxifen resistance were investigated by MTTassays, AnnexinV apoptosis assays and colony formation assay. Results: MCF-7TamRexosomes can enter into MCF-7wtrecipient cell. MCF-7TamRexosomes can increase theproliferation of MCF-7wtcells and decrease apoptosis upon tamoxifen treatment, thusenhancing tamoxifen resistance in recipient ER-positive breast cancer cells3. MCF-7TamRexosomes transport microRNA-221/222into recipient MCF-7wtcells.(1)The level of microRNA-221/222in MCF-7TamRand MCF-7wtcells and exosomes wastested by qPCR.(2) The level of microRNA-221/222in MCF-7wtcells treated withMCF-7TamRexosomes was tested by qPCR.(3) The level of p27and ERα in MCF-7wtcellstreated with MCF-7TamRexosomes was tested by WB. Results: Compared withMCF-7wtcells and exosomes, the level of microRNA-221/222in MCF-7TamRcells andexosomes was increased. MCF-7TamRexosomes can increase the level ofmicroRNA-221/222and decrease the levels of p27and ERα.4. Anti-miR-221/222blocks the propagation of tamoxifen resistance by exosomes.(1)qPCR and WB tested the efficiency of Anti-miR-221/222transfection.(2) The effect ofAnti-miR-221/222on tamoxifen resistance transimission was tested by the analysis of cellviability, apoptosis, and colony formation. Results: Anti-miR-221/222can increase thelevel of microRNA-221/222and decrease the levels of p27and ERα. Anti-miR-221/222can decrease the proliferation of MCF-7wtcells and increase apoptosis under tamoxifentreatment, thus inhibiting tamoxifen resistance in recipient ER-positive breast cancer cells.Conclusion(1) MCF-7TamRand MCF-7wtcells release exosomes with different characteristics.(2) MCF-7TamRexosomes can transfer tamoxifen resistance in ER postive breast cancer.(3) Tamoxifen resistance can be transferred by exosomal microRNA-221/222.