Quantitative proteomic analysis identifies novel regulators of methotrexate resistance in choriocarcinoma

Although methotrexate (MTX) is commonly used for the treatment of choriocarcinoma, chemoresistance to MTX may occur in a considerable fraction of patients. Further understanding on the mechanisms of MTX resistance would help to develop more effective therapy for choriocarcinoma.Quantitative proteomic approach involving TMT labeling and LC-MS/MS was used to identify MTX resistance-related proteomic profiles in choriocarcinoma cell models. Pathway and process enrichment analysis were conducted to identify MTX resistance-related biological processes/molecular pathways. CCK-8 viability assay, clonogenic survival assay, and BrdU incorporation analysis were used to examine the chemosensitivity to MTX in choriocarcinoma cells.In total, 5704 protein groups were identified, among which 4997 proteins were quantified. Bioinformatic analysis revealed that multiple biological processes/molecular pathways might be associated with MTX resistance in JEG3/JEG3/MTX cell systems. DPP4 and METTL7A were selected for further investigation. Increased expression of DPP4 or METTL7A was observed in MTX-resistant cancer cell lines and choriocarcinoma tissues. Knockdown of DPP4 or METTL7A significantly decreased cell viability, impaired clonogenesis, and increased apoptosis after MTX treatment in JEG3/MTX and JAR/MTX cells; while over-expression of DPP4 or METTL7A promoted cell viability and reduced apoptosis following exposure to MTX in JEG3, JAR and BEWO cells. Further, DPP4 and METTL7A differentially activated prosurvival signaling pathways including PI3K/AKT, ERK1/2 and STAT3, and attenuated the accumulation of reactive oxygen species (ROS) in choriocarcinoma cell lines.DPP4 and METTL7A might promote MTX resistance through activating pro-survival signaling pathways and attenuating the accumulation of ROS in choriocarcinoma cells. Targeting DPP4 and METTL7A might be useful to sensitize choriocarcinoma cells to MTX-based chemotherapy.