O-GlcNAc Modification Enhances Drug Resistance In Cancer Cells

Chemotherapy is one of the standard treatment methods for many cancers and development of chemoresistance,either intrinsic or acquired,is the most commonly encountered phenomenon that limits the success of cancer chemotherapy.Thus,investigating the molecular mechanisms of chemoresistance will provide new potential therapeutic targets for cancer therapy.Studies on the molecular mechanisms of tumor chemoresistance indicate that drug resistance is a regulation mode to adapt environmental changes,which is the result of a combination of various mechanisms.The mechanism of its occurrence needs to be further clarified.A growing body of evidence demonstrates that not only the abnormal expression of multidrug efflux transporters,such as P-glycoprotein(P-gp),but also apoptosis escape mediated by post-translational modification including protein glycosylation is one of the important mechanisms of drug resistance in tumor cells.Abnormal glycosylation is a common feature of cancer cells and is closely related to the biological behavior of tumors.Studies have shown that oxygen-linked acetyl glucosamine(O-GlcNAc)glycosylation is involved in cisplatin resistance in lung cancer cells.O-GlcNAcylation is a reversible monosaccharide modification catalyzed by O-GlcNAc glycosyltransferase(OGT),which exists in Ser/Thr residuces of cytoplasmic and nuclear protein.O-GlcNAcylation interacts with phosphorylation,which plays an important role in the regulation of numerous cellular physiological processes and major disease.However,the mechanism of this important glycosylation modification regulating cancer cells resistance remains to be clarified.In this study,by means of Western blotting,RT-PCR,Liquid chromatography-tandem MS(LC-MS/MS)and Annexin V–fluorescein isothiocyanate(FITC)/propidium iodide(PI)staining,the sensitive of chemotherapeutic and the changes of O-GlcNAcylation were analyzed.The relation between O-GlcNAcylation and chemoresistance is explained clearly and the new molecular mechanism how O-GlcNAcylation regulates chemoresistance.With DOX or CPT treatment,the report found that cells with intrinsic and acquired resistance were characterized by higher levels of O-GlcNAcylation than sensitive and parental cells.O-GlcNAcylated proteins were dynamically increased upon acute treatment with DOX and the expression of OGT had no significant changes,which suggests that the level of O-GlcNAc modification increases by DOX in an OGT-independent manner.Transfection of OGT siRNA inhibited the level of O-GlcNAc modification of intracellular proteins,which significantly reduced the drug resistance of tumor cells,indicating that O-GlcNAc glycosylation plays an important role in tumor cell chemoresistance.Furthermore,DOXstimulation up-regulated GFAT,a key enzyme of the aminohexose synthesis pathway(HBP),which expanded the flux through HBP,and then increases the accumulation of sugar donor UDP-GlcNAc required for O-GlcNAc glycosylation to induce protein O-GlcNAcylation.In conclusion,cell death induced by chemotherapy drugs were inhibited,which because GFAT increasing promoted protein O-GlcNAcylation.Through analyzing the molecular mechanism by which chemotherapeutic drugs up-regulated GFAT expression,we found that DOX treatment significantly increased AKT phosphorylation in drug-resistant cells(SMMC-7721 、 MCF-7/ADR),then XBP1 as downstream transcription factor was activated.XBP1 can directly regulate the transcription of GFAT.Using MK2206,the inhibitor of AKT,obviously decreased O-GlcNAc motification with the reductions in XBP1,GFAT expression and the content of intracellular UDP-GlcNAc and enhanced the sensitivity of drug-resistant cells to DOX.These results confirmed the chemotherapy agents activated the AKT/XBP1 axis and then induced the HBP.In addition,drug-induced O-GlcNAcylation could block the cleavage and activation of apoptotic and promote the phosphorylation of pro-survival transcription factors including NF-κB and AKT,then inhibited apoptotic.Meanwhile AKT activation enhanced HBP flux by a positive feedback mechanism,which further raised drug resistance in cancer cells.Since O-GlcNAcylation could regulate cancer cell chemoresistance,OSMI-1 as inhibitor of O-GlcNAcylation was used in combination with chemotherapeutic drugs to stimulate cancer cells and apoptotic was observed.OSMI-1 effectively sensitized DOX resistant SMMC-7721 and MCF-7/ADR cells to DOX and 5-FU.Consistent results were also observed in chemoresistant primary acute myeloid leukemia(AML)patient samples.Once again,that O-GlcNAcylation played an important role in chemoresistance was demonstrated.The combination of an O-GlcNAcylation inhibitor with chemotherapy could be a novel strategy in cancer therapy.In summary,the HBP has an important role in the cellular metabolic response to chemotherapy by regulating O-GlcNAcylation,and that this effect has important implications in drug resistance.First,we discovered that drug stress activates the HBP via the AKT/XBP1 axis.Second,O-GlcNAcylation is dynamically induced by the HBP and is required for cell survival and chemoresistance in cancer cells upon drug treatment.This post-translational modification contributes to the inhibition of apoptosis by blocking Caspase cleavage and the activation of prosurvival transcription factors,such as NF-κB and AKT,in response to chemotherapy.Furthermore,suppression of the HBP or O-GlcNAcylation reduces cancer cell resistance to chemotherapy.These results provide significant novel insights regarding the important role of the HBP and O-GlcNAcylation in regulating cancer chemoresistance.Thus,O-GlcNAc inhibition might offer a new strategy for improving the efficacy of chemotherapy.