Pancreatic ductal adenocarcinoma (PDAC) remains a highly fatal malignancy partially due to the acquired alterations related to aberrant protein glycosylation that pathologically remodel molecular biological processes and protect PDAC cells from death. Ferroptosis driven by lethal lipid peroxidation provides a targetable vulnerability for PDAC. However, the crosstalk between glycosylation and ferroptosis remains unclear. Here, we identified 4F2hc, a subunit of the glutamate-cystine antiporter system X_c^–, and its asparagine (N)-glycosylation is involved in PDAC ferroptosis by N- and O-linked glycoproteomics. Knockdown of SLC3A2 (gene name of 4F2hc) or blocking the N-glycosylation of 4F2hc potentiates ferroptosis sensitization of PDAC cells by impairing the activity of system X_c^– manifested by a marked decrease in intracellular glutathione. Mechanistically, we found that the glycosyltransferase B3GNT3 catalyzes the glycosylation of 4F2hc, stabilizes the 4F2hc protein, and enhances the interaction between 4F2hc and xCT. Knockout of B3GNT3 or deletion of enzymatically active B3GNT3 sensitizes PDAC cells to ferroptosis. Reconstitution of 4F2hc-deficient cells with wildtype 4F2hc restores ferroptosis resistance while glycosylation-mutated 4F2hc does not. Additionally, upon combination with a ferroptosis inducer, treatment with the classical N-glycosylation inhibitor tunicamycin (TM) markedly triggers the overactivation of lipid peroxidation and enhances the sensitivity of PDAC cells to ferroptosis. Notably, we confirmed that genetic perturbation of SLC3A2 or combination treatment with TM significantly augments ferroptosis-induced inhibition of orthotopic PDAC. Clinically, high expression of 4F2hc and B3GNT3 contributes to the progression and poor survival of PDAC patients. Collectively, our findings reveal a previously unappreciated function of N-glycosylation of 4F2hc in ferroptosis and suggest that dual targeting the vulnerabilities of N-glycosylation and ferroptosis may be an innovative therapeutic strategy for PDAC.

Background: There is an urgent need for the development of effective noninvasive biomarkers for early pancreatic cancer diagnosis. MicroRNAs (miRNAs) are promising candidates that can be identified in peripheral blood and can act as “liquid biopsy” biomarkers. miR-483-3p is overexpressed in the tumor tissue of pancreatic duct adenocarcinoma, but its potential as noninvasive biomarker remains unknown. Methods: We conducted locked nucleic acid in situ hybridization (LNA-ISH) for miR-483-3p in archival tissues of 107 patients with PDAC. We also used immunohistochemistry to evaluate SMAD4 expression, the putative miR-483-3p target gene. miR-483-3p expression level was also assessed using quantitative real-time PCR (qRT-PCR) in serum and serum exosome samples from 63 patients with PDAC and 22 healthy individuals. Results: LNA-ISH showed that miR-483-3p was overexpressed in PDAC and PanIN tissues compared to normal pancreatic duct cells. miR-483-3p expression levels correlated with increases in PanIN lesion grade. miR-483-3p expression negatively correlated with Smad4 expression (γ=− 0.770, p< 0.0001) in PDAC and PanIN tissues. Circulating miR-483-3p levels were significantly elevated in the serum and serum exosomes of PDAC patients compared to healthy controls (p< 0.0001 and p< 0.01, respectively). Specifically, serum miR-483-3p levels were able to distinguish patients with early stage (≤ 2cm) PDAC from healthy controls with an AUC of 0.83 [95% CI, 0.70– 0.96]. Higher serum exosomal miR-483-3p levels predicted worse survival in PDAC patients and serum exosomal miR-483-3p also proved to be an independent prognostic factor for PDAC (hazard ratio = 3.307; 95% CI=1.104 to 9.903; p=0.033). In vitro studies also showed that miR-483-3p promoted pancreatic cancer cell migration and invasion. Conclusion: miR-483-3p overexpression occurs early in PDAC development and is present in premalignant PanIN lesions. Serum miR-483-3p may act as an early PDAC diagnostic biomarker and serum exosomal miR-483-3p may be a PDAC prognostic biomarker.

Siglec‐15, a novel immune checkpoint, is an emerging target for next‐generation cancer immunotherapy. However, the role of Siglec‐15 in pancreatic ductal adenocarcinoma (PDAC) remains poorly understood. We investigated the expression of Siglec‐15 and its association with clinicopathological characteristics, programmed cell death‐ligand 1 (PD‐L1), immune cells, and DNA damage repair (DDR) molecules in a cohort of 291 patients with PDAC. Positive tumour cell expression of Siglec‐15 and PD‐L1 was observed in 18.6 and 30.3% of the samples, respectively. We also detected Siglec‐15 positivity in macrophages in 3.4% of patients. Co‐expression of Siglec‐15 with PD‐L1 was observed in 6.1% of the patients. A total of 33 PD‐L1‐negative samples (18.0%) were Siglec‐15‐positive. Siglec‐15 was observed more frequently in moderate‐to‐well‐differentiated tumours. Siglec‐15 was associated with a low density of Tregs and CD45RO T cells, high BRCA1 expression, and improved survival. Both Siglec‐15 and PD‐L1 are independent factors of patient outcomes. The prognostic significance of Siglec‐15 for survival was more discriminative in lymph node‐negative, high BRCA1 expression, or low BRCA2 expression tumours than in lymph node‐positive, low BRCA1 expression, or high BRCA2 expression tumours. In conclusion, we identified Siglec‐15 as a promising predictor for prognosis combined with different DDR molecular statuses and complex tumour‐infiltrating cells in PDAC. Targeting Siglec‐15 may be a novel therapeutic option for patients who are unresponsive to anti‐PD‐1 therapy. Future studies are needed to validate the prognostic significance of Siglec‐15 and to investigate its regulatory mechanisms in this disease.