Mesenchymal Stem Cells Drive Paclitaxel Resistance In ErbB2/ErbB3-Coexpressing Breast Cancer Cells And Its Underlying Mechanisms

Breast cancer is the most common malignancies in women worldwide,with the highest incidence rate and leading to more than 500,000 cancer deaths a year.ErbB2-overexpressing subtype is present approximately 25-30% of breast cancer with high risk and poor prognosis breast cancer.Paclitaxel is an important candidate drug for breast cancer chemotherapy.It is widely used against locally advanced and metastatic breast cancer,including in neoadjuvant treatment of ErbB2-positive breast cancer.With the extensive use of paclitaxel in chemotherapy,the innate and acquired resistance of paclitaxel has become increasingly problematic issue.However little is known about its related resistance mechanisms,which greatly hinders the therapeutic effect of breast cancer.Resistance to chemotherapeutics can be intrinsic or acquired.Intrinsic resistance means that resistance-mediating factors pre-exist in the context of tumor cells that make the therapy ineffective while patient receiving initial chemotherapy.Acquired drug resistance can develop during treatment of tumors and be caused by mutations arising during treatment,as well as through various other adaptive responses including activation of alternative compensatory signaling pathways.ErbB2-mediated drug resistance contributes to the malignancy of tumors and leading to lower survival of patients.However,there are no ligand has been found for ErbB2 so far.Its biological function was conducted by interacting with other receptors that can recognize the corresponding ligands.ErbB3 is often co-expressed in ErbB2-positive breast cancer cells and its expression level was closely related to ErbB2-induced tumor cell proliferation and survival.We have previously showed that elevated expression of ErbB3 results in paclitaxel resistance,and therapeutic targeting of ErbB3 enhances antitumor activity of paclitaxel against ErbB2-overexpressing breast cancer.The activation of downstream signal transduction pathways depends on the effective binding of ErbB3 to its ligand NRG-1,but it was found that the expression level of NRG-1 in ErbB2/ErbB3 co-expressing breast cancer cells is extremely low.In recent years,the role of tumor microenvironment in the biology of tumor has increasingly been recognized.Mesenchymal stem cells(also known as mesenchymal stromal cells,MSCs)can specifically recruit into many primary and metastatic tumor sites,including breast cancer.As an important component of tumor microenvironment(TME),MSCs may also play a critical role in regulating the proliferation and migration of cancer cell as well as the development of drug resistance via paracrine of exosomes with small active proteins,miRNAs,or lncRNAs.However,given the complex interaction between MSCs and tumor cells,it has been shown that MSCs may either promote or suppress tumor growth.Even contradictory results have been reported in different subtypes of breast cancer.Therefore,it is necessary to further investigate the effects of MSCs on specific subtype of breast canceras well as the underlying molecularmechanisms,clarifying the roleofmesenchymal stem cells as a large population of tumor microenvironment would help to further understand the machanisms of chemotherapy resistance in solid tumors.[Objective]This study aimed to explore the effects of MSC on proliferation and paclitaxel resistance in ErbB2/ErbB3-coexpressing breast cancer.The molecular mechanisms were further clarified to expand our understanding of cancer chemotherapy resistance and provide preliminary experimental evidence for targeted treatment in patients with ErbB2/ErbB3-coexpressing breast cancer.[Method]1.First,MSCs were isolated from Umbilical cord Wharton's jelly.Exosomes in MSC conditioned culture medium were enriched by ultrafiltration and were identified by transmission electron microscopy.The markers of exosomes were identified via WB and FACS.2.The effect of MSCs or its exosomes(0,1,2,5,10,20 ?g/ml)or rNRG1 on the proliferation of breast cancer MDA-MB-453 cells and the effect on the apoptosis of breast cancer cells induced by different concentrations of paclitaxel(0,2,4,8,16,32 nM)were assayed by MTS and ELISA,And then were verified by plate colony formation assay and FACS analysis.The mRNA expression of cyclinD1,cyclinE1 and cyclin dependent kinase inhibitors(p21,p27)were detected by RT-qPCR and WB.3.NRG-1 mRNA expression in MSCs and breast cancer cells was analyzed by PCR;the NRG-1 protein in MSC and its exosomes were detected by WB.The activation of Akt and MAPK signaling pathways and the expression of survivin and cleaved PARP in breast cancer cells were assayed after exosomes or NRG-1stimulated to investigate the molecular mechanisms of paclitaxel resistance.4.The pLKO.1_NRG-1_shRNA was constructed through Lentiviral vector.The expression of NRG-1 in MSCs and ErbB3 receptors in breast cancer cells were specifically knock down by NRG-1shRNA and ErbB3 shRNA,PI3K,AKT and Survivin breast cancer were inhibited by small molecule inhibitors LY294002,Akt inhibitor VIII and YM155,then the effects of MSC and its exosomes on breast cancer were re-analyzed by plate colony formation assay,MTS,ELISA and WB to verify the role of NRG1-ErbB3 mediated signaling pathway in MSC drive paclitaxel resistance in ErbB2/ErbB3 co-expression breast cancer.[Result]1.MSCs were isolated from the umbilical Wharton's jelly and were identified to meet MSC standards.The exosomes in MSC conditioned cultured medium were enriched by ultrafiltration and ultracentrifugation successfully.After identification,they are 30-100 nm in size and express the exosome markers such as CD9,CD63,and CD81,as well as umbilical cord MSC surface markers,which indicate that they are derived from Umbilical cord MSCs.2.MTS assay results showed that the effects of MSCs and its exosomes on proliferation of breast cancer MDA-MB-453 cells was not so obviously that the cell proliferation rates were little higher(116±2.53% and 114.5±0.48%,n=3);There was no statistical difference(p=0.061,p=0.092)compared with control group(100%).RT-qPCR results showed that the expression of CyclinD1,CyclinE1,p21,and p27 genes involved in cell cycle regulation in breast cancer MDA-MB-453 was 1.322±0.121,1.142±0.158,0.889±0.08 and 1.248±0.244 folds changed compared with control group;No significant changeswere found(p value=0.125,0.171,0.068 and 0.135).The WB results also confirmed that there was no significant change in protein level.3.Plate colony formation assay results showed that the number of breast cancer cells clones decreasing with the increase of paclitaxel(0,2,4,8 nM).While co-cultured with MSCs or recombinant NRG1 reduced the paclitaxel sensitivity of breast cancer MDA-MB-453 cells,and the cell clones were significantly increased compared with the group without MSC co-cultured.Quantitation results showed that breast cancer cells survival rates were(100±1.19%,85.69±1.04%,51.71±0.60%,18.88±0.62%)of control group,(104±1.24%,92.44±1.2%,75.29±1.2%,46.78±0.22%)of co-culture with MSCsgroup and(107±1.022%,94.24±0.67%,76.49±0.39%,40.69±0.37%)of NRG1 supplemented group.Thenumbers of breast cancer cellsafter treated with paclitaxel(2,4,8nM)were increased significantly in presence of MSCs(p=0.013,p=0.001,p=0.0001)or in presence of NRG1(p=0.0007,p=0.0003,p=0.0006).4.In paclitaxel-induced apoptosis of breast cancer cells model,breast cancer cells MDA-MB-453 survival rate decreasewith the increaseof paclitaxel(0,2,4,8,16,32 nM),while after supplemented with exosomes or rNRG1,breast cancer cell survival rate was significantly improved(p <0.01).Moreover,MSC exosomes reduced the paclitaxel sensitivity of breast cancer cells in a concentration-dependent manner(0,1,2,5,10,and 20 ?g/ml).When the exosomes concentration ismore than 2 ?g/ml(2,5,10,and 20 ?g/ml),the survival of breast cancer cells increased significantly(p=0.018,p=0.008,p=0.0003,and p=0.0001).5.Apoptosis analysis showed that the cytotoxicity of paclitaxel on breast cancer cells was inhibited significantly by MSC exosomes(p=0.0009);the cleaved PARP indicated apoptosis was reduced also.Flow cytometry analysis confirmed that supplemented with MSC exosome,the apoptosis rate of breast cancer cellsinducedby paclitaxel decreased from 43.2±0.71% to 23.35±0.64%,p<0.01).6.The results showed that NRG-1,the ErbB3 ligand,was expressed in MSC and its exosomes in high level,but not in breast cancer cells.MSC exosomes can significantly activate phosphorylation of ErbB3 and Akt in MDA-MB-453 breast cancer cells,and up regulated Survivin expression,while do not affect on the MAPK signaling.After NRG-1 of MSC was specifically knocked down by NRG-1shRNA,or the exosomes NRG-1 was neutralized by specific antibody,the effects were reversed.When ErbB3 was specifically knocked down,or PI3 K,AKT,Survivin signal path way was inhibited respectively,the effect of MSC and its exosomes on breast cancer cells was abolished.[Conclusion]1.Exosomes in MSCs conditioned culture medium could be enriched by centrifugal ultrafiltration-based method.2.MSC and its exosomes did not promote the proliferation of ErbB2/ErbB3 co-expressing breast cancer cells MDA-MB-453,but significantly reduced the sensitivity of breast cancer cell to chemotherapy drugs paclitaxel,induced paclitaxel resistance,and promoted the cancer cell survival.3.It was demonstrated that MSCs drive ErbB2/ErbB3 co-expressing breast cancer paclitaxel resistance by excreting NRG-1 cargoed by exosomes,which triggered activation of ErbB3/ErbB2 mediated AKT signaling cascade following survivin upregulation.4.Specific targeted inhibition of NRG-1 in MSCs or ErbB3,PI3K/AKT,Survivin in tumor cell can abolish the paclitaxel resistance of ErbB2/ErbB3 co-expressing breast cancer induced by MSCs.5.Our results suggest that targeting MSCs in the tumor microenvironment may be a novel strategy for overcoming paclitaxel resistance in ErbB2/ErbB3 co-expressing breast cancer patients.