Zhaohui Lu

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.

Assessing survival risk in patients with high-grade endometrial carcinomas has remained challenging. We aimed to investigate the distribution of molecular subtypes and assess their prognostic role in a large cohort of 355 patients with high-grade endometrial carcinoma. Molecular classification was determined using DNA polymerase epsilon (POLE) sequencing as well as immunohistochemical staining for p53 and mismatch repair (MMR) proteins. Endometrial carcinomas were stratified into four subtypes: POLE ultramutated, MMR-deficient, non-specific molecular profile (NSMP), and p53-mutant. This study included 177 and 178 patients with endometrioid and non-endometrioid carcinomas, respectively. Forty-two patients (11.8%) were categorized as POLE ultramutated, 106 (29.9%) as MMR-deficient, 128 (36.1%) as p53-mutant, and 79 (22.2%) as NSMP. Patients of different molecular subtypes had distinct survival times; molecular classification, but not histotype, was significantly associated with survival outcomes. When incorporating molecular classification into the stratification model, 52 patients (15.5%) switched risk groups, with 40 (11.9%) shifting to a lower risk for having a POLE mutation and 12 (3.6%) shifting to a higher risk owing to p53-mutant status. Molecular classification may provide more accurate prognostic information among patients with high-grade endometrial carcinomas and improve their stratification for purposes of clinical management.

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.

Immunotherapy targeting programmed cell death‐1 (PD‐1) has considerably improved the prognosis of patients with advanced cancers; however, its efficacy in the treatment of pancreatic ductal adenocarcinoma (PDAC) is unfavourable. To address the issue of PDAC immunotherapy, we investigated the expression of two PD‐1 ligands, PD‐L1 and PD‐L2, in PDAC, analysed their role in survival, and explored their correlation with clinicopathological features, immune infiltration, and DNA damage response molecules. Immunohistochemistry was performed on 291 surgically resected PDAC samples. In tumour cells (TCs) and immune cells (ICs), the positivity of PD‐L1 expression was 30 and 20% and that of PD‐L2 expression was 40 and 20%, respectively. Moreover, PD‐L1 expression on TCs correlated with its expression on ICs (p < 0.0001); a similar result was observed for PD‐L2 (p < 0.0001). Nonetheless, no correlation was observed between PD‐L1 and PD‐L2 expression. Positive PD‐L1 expression on TCs was related to N1 stage (p = 0.011) and AJCC II stage (p = 0.002), whereas positive PD‐L2 expression on TCs was associated with high FOXP3⁺ cell infiltration (p = 0.001) and high BRCA2 expression (p < 0.0001). Survival analysis revealed that positive PD‐L1 (p = 0.046) and PD‐L2 (p = 0.028) expression on TCs was an independent risk factor for unfavourable disease‐specific survival (DSS). Furthermore, positive PD‐L2 expression on TCs was an independent risk factor for lower DSS in the pN0 (p = 0.023), moderate and well tumour differentiation (p = 0.004), low BRCA1 (p = 0.017), wild‐type p53 (p = 0.034), and proficient mismatch repair (p = 0.004) subgroups. Moreover, post‐operative adjuvant chemotherapy could significantly affect DSS, regardless of PD‐L1/PD‐L2 expression status (positive or negative) on TCs, while it only prolonged DSS in PDL1‐ICs⁽⁻⁾ (p < 0.0001) and PDL2‐ICs⁽⁻⁾ (p < 0.0001) subgroups. This study provides a comprehensive understanding of the roles of PD‐L1 and PD‐L2 in PDAC, supporting anti‐PD‐1 axis immunotherapy for PDAC.

Yu S, et al. J Gynecol Oncol. 2022 Feb;33(3):e38. https://doi.org/10.3802/jgo.2022.33.e38

Colorectal cancer (CRC) is the third most common malignancy worldwide. The novel immune checkpoint V-domain Ig suppressor of T-cell activation (VISTA) has emerged as a promising target for cancer treatment; however, the prognostic significance of its expression in CRC remains unknown. In this study, immunohistochemical staining was used to investigate VISTA expression in tissue microarrays from 1434 patients with stage I–III CRC (816 in the exploratory cohort and 618 in the validation cohort). VISTA protein was evaluated separately in tumor cells and tumor-infiltrating immune cells (ICs). The associations between VISTA expression, mismatch repair (MMR) status, and clinicopathologic parameters were analyzed, as was the effect of VISTA on survival. High VISTA expression on ICs (ie, ≥5% staining) was more frequent in patients with N0 stage, T1–2 stage, low tumor grade, high CD8 density, and MMR-deficient tumors, and was positively associated with prolonged survival in patients with CRC. High VISTA expression was a significant predictor of prolonged survival independent of clinicopathologic parameters and MMR status. Overall, our results indicate that high VISTA expression on tumor-infiltrating ICs correlates with early tumor stage, MMR deficiency, and a favorable prognosis in patients with CRC. This ought to be considered in future trials of VISTA-modulating immunotherapy for patients with CRC.

Objective: The identification of DNA polymerase epsilon (POLE) mutation subtypes in endometrial cancer is critical for molecular classification. The mutation of the POLE gene could only be detected by sequencing until now. We propose to validate and develop the feasibility of using BaseScope, an in situ hybridization (ISH) assay, for the detection of POLE mutations in high-grade endometrioid carcinomas (EC). Methods: Among 51 paraffin-embedded samples of high-grade EC, BaseScope-ISH assays were used to detect the RNA mutation status of the POLE gene, mainly focusing on two hotspot mutations of P286R and V411L. The number of positive signals in the cytoplasm was counted, setting the positive threshold and determining the in situ hybridization results. The sensitivity and specificity of BaseScope-ISH assay were compared with that of the Sanger sequencing results. Results: Based on the BaseScope assay, there were 19 positive samples and 32 negative samples in a total of 51 samples. Of the 19 positive samples, 10 samples showed P286R site mutations in the POLE gene, while the other nine samples were V411L site mutations. Only one sample with the V411L site mutation identified by Sanger sequencing showed negative signal value. The remaining 31 cases without the P286R site mutation or V411L site mutations all showed negative signal. This analysis result showed the sensitivity was 95% and the specificity was 100% for the BaseScope assay detecting POLE mutants in high-grade EC. Conclusion: In the case of high-grade EC, combined with morphological characteristics, the BaseScope assay can effectively and specifically identify POLE mutation cases, providing a reliable foundation for the application of clinical diagnosis and molecular classification.

Purpose This study aimed to examine whether the different tumor-transplanted sites could construct a similar immunoinflammatory microenvironment and to investigate the interactions between tumor microenvironment cells. Methods The red fluorescent protein-SU3 (SU3-RFP) or SU3 glioma stem cells (GSC) were inoculated into the brain, liver, abdominal cavity, and subcutis of green fluorescent protein (GFP)-nude mice. The tumor tissues were taken to observe the tissue cell distribution. The single cell suspension of tumor tissues was prepared and cultured, while the SU3-RFP cells were co-cultured with the cells from GFP-transgenic mice. The RFP⁺, GFP⁺, and RFP⁺/GFP⁺ cells were traced by fluorescence microscope, and their protein expressions were determined by Western blot analysis. The markers of immunoinflammatory cells, including F4/80, CD11b, CD11c, CD80, CD47, and SIRP-α, were determined by RT-PCR and immunocytochemistry assays, respectively. Results The xenograft models of all transplant sites were inducible, and the red tumor cells of tumor tissues were encircled by a great quantity of host-derived green cells, including immunoinflammatory cells with CD80, F4/80, CD11b, and CD11c expressions, which might generate the cell colonies and possess the pseudopodia. Additionally, the interactions between red tumor cells and green immunoinflammatory cells, including cell fusion process and yellow fusion cell formation, were observed in cultured cells. The fusion cells-derived B4 cells with expressions of CD47 and SIRP-α proteins had the strong proliferation ability and tumorigenic effect. Conclusions The similar tumor immunoinflammatory microenvironment was constructed by GSC in different transplant sites, and the cell fusion indicated a malignant transformation of the tumor microenvironment cells.