| Pancreatic ductal adenocarcinoma(PDAC)is a devastating human malignancy with an average 5-year survival rate less than 8%.Due to its hidden anatomical site,atypical clinical symptoms,and low specificity of tumor markers,most PDAC patients are diagnosed with advanced disease,which are not suitable for surgical resection.Chemotherapy is the first choice for its treatment,among which gemcitabine represents the first-line treatment of PDAC,but drug resistance is a major obstacle in improving t he patient’s response which eventually leads to chemotherapy failure.Therefore,analyzing and revealing the molecular basis of regulating pancreatic cancer chemoresistance will provide corresponding theoretical and experimental basis for pancreatic cancer treatment.Recently,exosomes have emerged as important mediators for cell-to-cell communication.Exosomes engulf biologically active cargos including proteins,RNAs and lipids,which can be uptaken by adjacent cells and affect their behavior.In addition,exosomes shed from tumor microenvironment were found to promote the stemness,epithelial-mesenchymal transition(EMT),metastasis and chemotherapy resistance of cancer cell s.Pancreatic cancer cells have a strong secretory function,and secreted proteins play an important role in the process of intercellular information transmission.Therefore,the pancreatic cancer drug-resistant cells Bx PC-3-Gem obtained through long-term gemcitabine screening and its parental line Bx PC-3were used as models to investigate the secretome changes of pancreatic cancer drug-resistant cells through protein profiling analysis.The results showed that the secreted proteins in the conditioned medium from drug-resistant cells were upregulated in relative to those from the sensiti ve cells.The different level of MMP14 between drug-resistant Bx PC-3-Gem cells and sensitive parental cells was extremely high.MMP14 was most closely related to the regulation of EMT,tumor stemness and other functions by GO analysis.GSEA and the network analysis of secreted proteins confirmed that there are more secreted protein components related to plasma membrane system,tumor stemness and EMT in drug-resistant Bx PC-3-Gem cells.The enhanced tumor stemness is considered to be the root cause of chemotherapy resistance in pancreatic cancer.Therefore,these results suggest that MMP14 is a key protein s ecreted by drug-resistant cells involved in drug resistance regulation.It functions as a drug-resistant acquired protein in pancreatic cancer.MMP14 is a transmembrane protein,and its presence in conditioned medium of pancreatic cancer cells suggests tha t MMP14 may be secreted in the exosomes.To verify the above hypothesis,the existence of MMP14 in exosomes was confirmed,and it was confirmed that MMP14 in exosomes secreted by drug-resistant cells could be absorbed by recipient cells.The function of MMP14 in tumor stem cells and chemotherapy resistance was analyzed through the overexpression and silencing experiments of MMP14.The relationship between gemcitabine drug resistance and exosomal MMP14 was demonstrated by co-culture experiments of resistant cells and sensitive cells.The results showed that the exosome MMP14 derived from drug-resistant cells could regulate the EMT process,stemness and drug resistance of sensitive cells.Exosomes with high content of MMP14 can enhance the s temness,invasion and metastasis of recipient cells,thus transmitting drug resistance from drugresistant cells to sensitive cellsCD44 is an important marker that regulates the properties of tumor stem cells.In this study,we found that CD44 protein lev els in recipient cells were upregulated after exosome treatment.Since MMP14 can bind to CD44,this study investigated the regulatory effect of exosome MMP14 on recipient cel l CD44.The results showed that MMP14 had no effect on its m RNA expression level,but influenced its protein level.Further research revealed that MMP14 could stabilize CD44 protein,causing the acquisition of stemness characteristics of sensitive pancreatic cancer cells.Taken together,this study elucidates the role of drug-resistant cells through exosome-transffered MMP14 to transmit tumor stemness phenotype characteristics and affect the chemoresistance of the surrounding PDAC sensitive cells.Our results indicate that MMP14 is a key player for exosome-mediated transfer of gemcitabine resistance,and reveal the molecular mechanism by which exosome-transffered MMP14 transmits resistance by enhancing the stability of CD44 in recipient cells.It provides a theoretical and experimental basis for targeting exosome-transferred MMP14 to improve the sensitivity of pancreatic cancer to gemcitabine chemotherapy. |
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