Effect And Mechanism Of 4F2hc N-glycosylation Mediated By Glycosyltransferase B3GNT3 On Ferroptosis In Pancreatic Ductal Adenocarcinom

Background:Pancreatic ductal adenocarcinoma(PDAC)is the most common aggressive pancreatic malignancy,accounts for more than 90%of pancreatic cancer cases,and remains a clinically lethal cancer with a 5-year relative survival rate of approximately 11%.Current multiple-treatment strategies to slow or prevent PDAC exacerbation,include surgical resection,adjuvant chemotherapy(ACT),and immunotherapy.Regrettably,the overall response rate remains poor for most PDAC patients.Therefore,we urgently need to advance our understanding of the mechanism of PDAC biology and develop alternative multimodal therapeutic options for the eradication of PDAC.Aberrant glycosylation involves multifaceted pathological and pathophysiological changes in PDAC,including but not limited to conferring tumor cells the ability to resist cell death.Glycosylation,a widespread posttranslational modification(PTM),plays a significant role in stabilizing protein structure,guides proteins to move to the right location,and helps molecular chaperones to fold properly.Emerging evidence indicates that aberrant protein glycosylation can pathologically remodel molecular biological processes and protect PDAC cells from death.Ferroptosis is a novel form of regulated cell death characterized by the iron-dependent unrestricted toxic accumulation of lipid peroxidation products and plasma membrane rupture,it is a novel and effective method for PDAC eradication.Among the numerous regulatory pathways involved in ferroptosis defense,the glutamate-cystine antiporter system Xc-is widely known and consists of two subunits,namely,the heavy chain subunit 4F2hc(also known as CD98hc,encoded by the SLC3A2 gene)and the light chain subunit xCT(encoded by the SLC7A11 gene),which mediate the synthesis of cysteinederived antioxidants and the following establishment of the anti-oxidation system.Both glycosylation and ferroptosis are physiological metabolic processes that may provide new therapeutic opportunities for cancer treatment.However,hitherto the crosstalk between glycosylation and ferroptosis has not yet been largely revealed,and whether targeting the glycosylation process can enhance PDAC to ferroptosis sensitivity.Methods:1.To reveal the changes and specific types of glycosylation modification during PDAC ferroptosis,finding the differentially expressed glycoproteins and glycopeptides involved in the regulation of PDAC ferroptosis,and identifying the potential glycoproteins associated with ferroptosis,N-/O-glycoproteomics were performed in this study.2.To find out which type of glycosylation is critical for the modification of target glycoproteins,N-glycosidase(PNGase F)(a recombinant glycosidase mainly used to remove oligosaccharides connected to asparagine residues on peptide chains)or protein Nglycosylation inhibitor tunicamycin(TM)were used in this part.3.RNA-seq technology was used to explore the changes of glycosyltransferases during PDAC ferroptosis and identify potential differentially expressed glycosyltransferases responsible for target glycoprotein.4.291 archived samples from PDAC patients and their detailed clinicopathological data and prognostic information were used to investigate the expression and clinical prognostic significance of glycoprotein 4F2hc and glycosyltransferase B3GNT3.5.Lentivirus-mediated SLC3A2-specific stable knockdown cell lines were used to explore the specific role of 4F2hc in PDAC ferroptosis and determine the impact of N-glycosylation modification on PDAC ferroptosis.6.Lentivirus-packaged CRISPR-cas9-mediated gene knockout technology was used to explore the specific role of B3GNT3 and its glycosyltransferase activity in PDAC ferroptosis.7.Amino acid site mutation,fragment truncation,molecular dynamics simulation,DynaMut,and PredyFlexy for predicting protein stability,immunofluorescence,flow cytometry,and cycloheximide chase assay were used to investigate the impact of N-glycosylation modification and the glycosyltransferase activity of B3GNT3 on 4F2hc protein stability and the effect of inhibiting Nglycosylation of 4F2hc on cell membrane localization,expression,and function of its molecular partner xCT.8.PDAC subcutaneous and orthotopic transplantation tumor models were established to investigate the impact of 4F2hc and B3GNT3 on the proliferation activity of PDAC cells in vivo,Genetic perturbation of SLC3A2 combined with ferroptosis inducer IKE or protein Nglycosylation inhibitor tunicamycin were used to explore the proliferation activity of orthotopic PDAC.Results:1.The levels of N-glycosylation were significantly increased during PDAC cell ferroptosis,the N-glycosylation of differentially upregulated glycoprotein 4F2hc may affect the process of PDAC cell ferroptosis.2.N-glycosylation is the main post-transcriptional modification of 4F2hc protein,we identified B3GNT3,but not other glucosyltransferases,as a bona fide binding partner for 4F2hc protein and may be involved in N-glycosylation of 4F2hc.3.Both 4F2hc and B3GNT3 are highly expressed in PD AC cells and clinical PD AC tissues.Notably,high expression of 4F2hc and B3GNT3 contributes to the progression and poor survival of PD AC patients.4.Mechanistically,the knockdown of SLC3A2(gene name of 4F2hc)or blocking of the Nglycosylation of 4F2hc potentiates ferroptosis sensitization of PD AC cells by impairing the activity of system Xc-(manifested by a marked increase of lipid peroxidation).Reconstitution of wildtype SLC3A2 exhibited a much more pronounced protective effect against RSL3induced ferroptosis in shSLC3A2 PANC-1 and MIA PaCa-2 cells.While N365Q and 4NQ mutants failed to restore the cell viability and showed greater sensitivity to RSL3-induced ferroptosis.5.Gene knockdown or knockout of B3GNT3 sensitizes PD AC cells to ferroptosis.Reconstitution of enzymatically active 122-311OE partially restored the cell viability in B3GNT3KO PANC-1 and MIA PaCa-2 cells treated with RSL3,while reconstitution with a glycosyltransferase-null 122-31del constructs could not.6.Knockout of B3GNT3 accelerates the protein degradation of 4F2hc and decreases its cell membrane localization and interaction with xCT.Regardless of whether mutate the Nglycosylation sites of 4F2hc or use of tunicamycin to remove the N-glycan structure of 4F2hc,it significantly reduces the Gibbs free energy(ΔΔG)of 4F2hc protein,and leads to the instability of 4F2hc protein and weakens its interaction with xCT.7.The N-glycosylation inhibitor TM significantly enhances lipid peroxidation and ferroptosis induced by IKE and RSL3 in PDAC cells.8.In in vivo studies,knockout of B3GNT3 significantly inhibits subcutaneous and orthotopic transplantation tumor growth of PANC-1 cells.Genetic perturbation of SLC3A2 or combination treatment with TM markedly augments ferroptosis-induced inhibition of orthotopic PDAC.Conclusion:1.N-glycosylation is involved in the process of PDAC ferroptosis.2.The N-glycosylation modification of 4F2hc mediated by glycosyltransferase B3GNT3 affects the sensitivity of PDAC cells to ferroptosis.3,Targeting the B3GNT3-4F2hc-N-glycosylation pathway leads to the reduction of the stability and membrane localization of 4F2hc protein,and disrupts the interaction between 4F2hc and xCT,thereby promoting the sensitivity of PDAC to ferroptosis.4.High expression of 4F2hc and B3GNT3 in PDAC predicts poor clinical prognosis for patients,and inhibiting their expression can effectively reduce the proliferative activity of PDAC cells in vitro and in vivo.Significance:1.This study aims to uncover the potential regulatory mechanism of ferroptosis-induced cell death in PDAC cells through a novel perspective of glycosylation.2.The integration of N-/O-glycosylation proteomics and transcriptomics will provide a critical research approach to deciphering changes in glycosylation modification patterns during PDAC ferroptosis.3.By examining the differential changes in glycosylation sites,we can screen and identify the critical sites that respond to differential glycoproteins in the ferroptosis regulatory pathway,providing novel therapeutic targets and potential biomarkers for the treatment of malignant tumors based on ferroptosis strategies.4.This study seeks to lay an experimental foundation for examining the therapeutic potential of glycosylation inhibitors and ferroptosis inducers in preclinical research for the treatment of malignant tumors.5.Based on the newly discovered regulatory role of the B3GNT3-4F2hc-N-glycosylation axis in ferroptosis,this study provides novel research direction for expanding and developing the clinical treatment strategies for anti-cancer purposes.