Beyond antibody fucosylation: α-(1,6)-fucosyltransferase (Fut8) as a potential new therapeutic target for cancer immunotherapy

[This retracts the article DOI: 10.1155/2022/2271788.].

Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) is an E3 ubiquitin ligase and scaffolding protein that contribute to the progression of various malignant tumors. However, the role of TRAF2 expression in epigenetic, cancer prognosis, and immune responses in tumor microenvironment is unclear. We used The Human Protein Atlas (HPA) database, TIMER 2.0 database, and TCGA database to evaluate TRAF2 expression in human normal and tumor tissues. Correlation of TRAF2 expression with mutations and epigenetic in tumors was evaluated using the cBioPortal platform and the GSCA database. To assess the prognostic value of TRAF2, we performed Kaplan-Meier plots and Cox regression analysis. LinkedOmics database was used for PANTHER Pathways enrichment analysis. The relationship between TRAF2 expression and immune checkpoint genes, as well as immune cell infiltration, was examined using TIMER 2.0 and the R language. Single-cell sequencing data and multiple immunofluorescence staining were used to observe the co-expression of TRAF2 on hepatocellular carcinoma cells and immune cells. Furthermore, using siRNA-mediated knockdown, we explored the potential role of TRAF2 in liver cancer cell biology. Our findings indicate that TRAF2 is frequently mutated and significantly overexpressed in various types of cancers, and this overexpression is linked to a poor prognosis. The epigenetic alterations in TRAF2 was significant across various types of cancers. TRAF2 is associated with the levels of various immune checkpoint genes and multiple tumor-infiltrating immune cells, suggesting its potential involvement in tumor microenvironment. Of note, enrichment analysis revealed a significant correlation between TRAF2 and T cell activation, and single-cell sequencing indicated that TRAF2 was overexpressed in malignant cells and T cells. In vivo results demonstrated that TRAF2 was closely associated with T lymphocytes in hepatocellular carcinoma. The results of our in vitro experimental studies confirmed that the loss of TRAF2 function inhibits the malignant behavior of HepG2 cells in hepatocellular carcinoma. TRAF2 represents a potential prognostic biomarker and therapeutic target for cancer immunotherapy, particularly in patients with hepatocellular carcinoma.

<div>Abstract<p>Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein–coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1–4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4<sup>fl/fl</sup>Lyz2<sup>cre/+</sup> mice, characterized by fewer protumoral M2 TAMs and increased CD8<sup>+</sup> T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy.</p><p><b>Significance:</b> This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy–resistant lung cancer to anti-PD-1 therapy.</p><p><b>Graphical Abstract:</b> <a href="http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg" target="_blank">http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg</a>. <i>Cancer Res; 78(17); 4929–42. ©2018 AACR</i>.</p></div>

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous family of immune cells including granulocytic (CD14neg/CD15+/HLA-DRneg) and monocytic subtypes (CD14+/CD15neg/HLA-DRneg). In the present study, we found a population of monocytes expressing the granulocyte marker CD15 that significantly increased in both peripheral blood (PB) and tumoral tissues of patients with colorectal cancer (CRC). Further phenotypical analysis confirmed the granulocytic-like features of this monocyte subpopulation that is associated with an increase in granulocyte-monocyte precursors (GMPs) in the PB of these patients (pts). Mechanistically, this granulocyte-like monocyte population suppressed NK cell activity by inducing TIGIT and engaging NKp30. Accordingly, an increased frequency of TIGIT+ NK cells with impaired functions was found in both the PB and tumoral tissue of CRC pts. Collectively, we provided new mechanistic explanations for tumor immune escape occurring in CRC by showing the increase in this new kind of MDSC, in both PB and CRC tissue, which is able to significantly impair the effector functions of NK cells, thereby representing a potential therapeutic target for cancer immunotherapy.

Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein-coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1-4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4fl/flLyz2cre/+ mice, characterized by fewer protumoral M2 TAMs and increased CD8+ T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy.Significance: This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy-resistant lung cancer to anti-PD-1 therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg Cancer Res; 78(17); 4929-42. ©2018 AACR.

Background: Abnormal activation of endoplasmic reticulum (ER) stress sensors and their downstream signalling pathways is a key regulator of tumour growth, tumour metastasis and the response to chemotherapy, targeted therapy and immunotherapy. However, the study of ER stress on the immune microenvironment of bladder urothelial carcinoma (BLCA) is still insufficient. Methods: Firstly, 23 ER stress genes were selected to analyse their expression differences and prognostic value in BLCA based on the existing BLCA genome atlas data. According to the expression level of ER stress-related genes in BLCA, two independent clusters were identified using consensus cluster analysis. Subsequently, the correlation between these two clusters in terms of the immune microenvironment and their prognostic value was analysed. Finally, we analysed the prognostic value of the key ER stress gene HSP90B1 in BLCA and its corresponding mechanism that affects the immune microenvironment. Results: Consensus clustering showed a worse prognosis and higher expression of immunoassay site-related genes (HAVCR2, PDCD1, CTLA4, CD274, LAG3, TIGIT and PDCD1LG2) in cluster 1 compared with cluster 2. Additionally, both TIMER and CIBERSORT algorithms showed that the expression of immune infiltrating cells in cluster 1 was significantly higher than that in cluster 2. Subsequently, HSP90B1 was identified as a key ER stress gene in BLCA, and its high expression indicated poor prognosis and was closely related to PD1. We also analysed the correlation between HSP90B1 expression and immune-infiltrating cell related biomarkers, which showed positive results. Finally, we verified the prognostic value of HSP90B1 in BLCA using an immunohistochemical assay in a tissue microarray of 100 patients with BLCA, validating the potential of HSP90B1 as a prognostic biomarker in patients with BLCA. Conclusion: Our work reveals that ER stress genes play a crucial role in the BLCA immunological milieu, and HSP90B1 is a potential prognostic biomarker and therapeutic target for cancer immunotherapy.

PANoptosis, an integrated form of programmed cell death encompassing pyroptosis, apoptosis, and necroptosis, has emerged as a key mechanism at the intersection of inflammation and immunity [1] . Acting through the PANoptosome complex, this multifaceted pathway not only regulates cellular homeostasis but also orchestrates immune activation and tissue remodeling. In the tumor immune microenvironment, PANoptosis plays dual roles: it can amplify anti-tumor immunity or, conversely, induce immune exhaustion and immune escape when dysregulated [2,3] . T cell-based immunotherapy-including immune checkpoint blockade (ICB) and chimeric antigen receptor (CAR) T cell therapy-has transformed the therapeutic landscape of multiple cancers [4] . However, the variability in patient responses underscores the complexity of immune regulation and cell death signaling. Exploring how PANoptosis modulates T cell activity offers new insights into the mechanisms underlying immunotherapy resistance and tumor immune evasion. This Research Topic gathers a series of studies that collectively explore this dynamic interplay from molecular discovery to translational potential.At the molecular level, Wang et al. applied a multi-omics strategy to classify renal cell carcinoma (RCC) into distinct metabolic subtypes. Their findings revealed that metabolic reprogramming tightly associates with PANoptosis-related signaling, defining prognostic subgroups and potential targets for precision immunotherapy. Building on the theme of molecular regulation, Tao et al. investigated cathepsin Z (CTSZ) in prostate cancer and found that its overexpression not only predicts poor clinical outcomes but also shapes an immunosuppressive microenvironment characterized by elevated PD-1 and PD-L1 expression-linking protease signaling to checkpoint regulation and PANoptotic control.Expanding this exploration into other cancer types, Lei et al. developed a programmed cell death (PCD) score model for hepatocellular carcinoma (HCC) that integrates transcriptomic and single-cell analyses. Their identification of UBE2E1 as a key oncogenic driver associated with high PCD scores highlights how cell death dynamics affect T cell exhaustion and tumor aggressiveness. Complementing this, Wu et al. conducted a pancancer analysis of PLAG1 and demonstrated its functional role in promoting tumor proliferation and immune evasion in bladder cancer, further strengthening the connection between PANoptosis and immune modulation.Moving from genomics to the tumor microenvironment, Liao et al. explored the clinical relevance of tumor mutation burden (TMB) across cancers and identified RPLP0 as a predictive biomarker for immunotherapy response. Interestingly, RPLP0 knockdown enhanced anti-PD-L1 efficacy in bladder cancer models, providing a tangible example of how PANoptosis-related translational control may affect therapeutic sensitivity. Similarly, Wan et al. synthesized current findings on immune cell PANoptosis in colorectal cancer, emphasizing that excessive immune cell death can undermine anti-tumor immunity and proposing strategies to restore immune balance through targeted interventions.Beyond oncology, Chen et al. extended the concept of PANoptosis to diabetic retinopathy, identifying several hub genes that regulate inflammation and immune cell infiltration. Their integrative machine-learning approach underscores that PANoptotic signaling has broad implications in immune-related diseases beyond cancer. In parallel, Ma et al. reviewed advances in immunogenic cell death (ICD) and nanomaterial-based therapies, highlighting how nanotechnology can trigger PANoptosis-like mechanisms to promote antigen release and potentiate durable T cell immunity. These findings demonstrate that manipulating cell death pathways can synergize with T cell-based therapies to achieve improved clinical outcomes.Together, the articles in this Research Topic illuminate PANoptosis as a pivotal immunoregulatory hub bridging cell death, metabolism, and immune activation. They reveal how fine-tuning PANoptotic signaling can shape tumor immunogenicity, determine therapeutic response, and open new avenues for combinatorial immunotherapy. Future studies integrating single-cell, spatial, and multi-omics technologies will be essential to define the spatial and temporal coordination of PANoptosis within the tumor microenvironment. By deepening our mechanistic understanding, researchers may unlock strategies to exploit PANoptosis for enhanced T cell function, reinvigorated anti-tumor immunity, and ultimately improved patient survival.

Pancreatic cancer (PC) remains a major health concern, with conventional cancer treatments exerting little influence on the disease course. MicroRNA-520b (miR-520b) functions as a tumor suppressor in several types of human cancers, whereas its anti-tumor property in the context of PC is still fundamental. The aim of this study is to identify the potential therapeutic role of miR-520b, transferred by exosomes, derived from normal fibroblasts (NFs) in PC progression. A gain-of-function study was performed to examine the roles of miR-520b in PC cell line SW1990, which suggested that miR-520b served as a tumor suppressor in PC. In order to confirm the role of exosomal miR-520b, exosomes were isolated from NF culture medium and cocultured with SW1990 cells. During the coculture experiments, we disrupted exosome secretion and upregulated exosomal miR-520b. The in vitro coculture studies revealed that miR-520b was transferred from NF-derived exosomes to PC cells and thereby suppressed PC cell proliferation, invasion, migration, and stimulated apoptosis. Furthermore, inhibited tumor growth and live metastasis upon elevated miR-520b in exosomes were observed in vivo. Conjointly, our study demonstrates that NF-derived exosomal miR-520b impedes the progression of PC, which contributes to a novel, therapeutic role of exosomal miR-520b for treating PC.

Long non-coding RNA NEAT1 facilitates pancreatic cancer progression through negative modulation of miR-506-3p

Ras proteins regulate multiple phenotypes, including proliferation, contact inhibition, cell motility, metabolism, and genome integrity. This range of phenotypes may relate to the number of different effector pathways that Ras activates. The best validated of these is the Raf/MAPK pathway. Ras proteins can activate PI 3-kinase pathways directly, though this seems to vary between tissue types. Ras proteins bind and activate RalGDS, but this pathway is less well understood. In addition to these three major pathways, Ras proteins in their GTP state interact directly with several other potential effectors. Analysis of the contributions that these pathways make towards cancer phenotypes using genetic approaches or RNA interference has been complicated by redundancies within each effector pathway. For example, there are three Raf isoforms, 3 PI 3-kinase isoforms, etc. This explains why screens for single genes that Ras depends on have not identified classical Ras effectors. Furthermore, each pathway may have several relevant phenotypes, such as effects on cell cycle progression, survival, and metabolic stress. Therefore, screens that depend on one phenotype, such as proliferation or survival, might miss other important functions. In addition to these technical complications, it is important to recognize differences in signaling between different tumor types. For example, in lung adenocarcinoma, the RTK/RAS/MAPK pathway can be activated by mutations in RTKs, activation of SOS, loss of NF1, mutation in KRAS, or activation of BRAF, with similar clinical outcomes. In pancreatic cancer, only mutation in KRAS appears capable of driving malignancy. In lung adenocarcinoma and pancreatic cancer, KRAS mutations are likely initiating events, in colorectal cancer they are definitely not, and exist in the context of un-regulated beta-catenin signaling. To address these issues we analyzed the importance of each canonical effector pathway, plus 37 other potential effectors, in 100 cell lines derived from lung, pancreatic and GI tissues, measuring five different parameters on a single cell basis. We used combinations of highly potent and well characterized siRNAs that enabled us to knock down all the paralogs within an effector node at once. For example, we were able to knock down ARAF, BRAF and RAF1, together, in the “RAF node”. We took advantage of the variable nature of siRNA knockdown to generate dose-response curves for each node, measuring effects on five parameters on each cell. This analysis revealed the expected dependence on tissue of origin, with pancreatic cells being more dependent on Ras signaling than lung adenocarcinoma. Unexpectedly, we found that most KRAS mutant tumor cell lines fell into two major classes: those that depend strongly on KRAS itself, and the Raf/MAPK pathway, and those that had lost dependence on KRAS, but were strongly dependent on RSK p90 S6 kinase. These cells were not dependent on PI 3-kinase genes themselves, but rather on PDK1, loss of LKB1 and other aspects of downstream PI 3-kinase signaling. We also identified a small number of cells that depended strongly on RalGDS. Each of these subsets had remarkably different properties, in terms of energy metabolism, EMT status, and cell-cell interaction. We conclude that oncogenic Ras proteins can indeed activate several downstream pathways, but in individual cell lines, one effector pathway predominates. Citation Format: Tina Yuan, Frank McCormick. Regulation of Ras proteins and their effectors. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA05.

Identification of epigenetically silenced genes in human pancreatic cancer by a novel method “microarray coupled with methyl-CpG targeted transcriptional activation” (MeTA-array)

Cancer stem cells (CSCs), which are found in various types of human cancer, including pancreatic cancer, possess elevated metastatic potential, lead to tumor recurrence and cause chemoradiotherapy resistance. Alterations in cellular bioenergetics through the regulation of 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling may be a prerequisite to stemness. Betulinic acid (BA) is a well-known bioactive compound with antiretroviral and anti-inflammatory potential, which has been reported to exert anticancer effects on various types of cancer, including pancreatic cancer. The present study aimed to investigate whether BA could inhibit pancreatic CSCs via regulation of AMPK signaling. The proliferation of pancreatic cancer cells was examined by MTT and colony formation assays. The migratory and invasive abilities of pancreatic cancer cells were assessed using wound-scratch and Transwell invasion assays. In addition, the expression levels of candidate genes were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. The results revealed that BA inhibited the proliferation and tumorsphere formation of pancreatic cancer cells, suppressed epithelial-mesenchymal transition (EMT), migration and invasion, and reduced the expression of three pluripotency factors [SRY-box 2 (Sox2), octamer-binding protein 4 (Oct4) and Nanog]. Furthermore, immunohistochemical analysis confirmed that there was a significant inverse association between the expression levels of phosphorylated (P)-AMPK and Sox2 in pancreatic cancer, and it was revealed that BA may activate AMPK signaling. Notably, knockdown of AMPK reversed the suppressive effects of BA on EMT and stemness of pancreatic cancer cells. In addition, BA reversed the effects of gemcitabine on stemness and enhanced the sensitivity of pancreatic cancer cells to gemcitabine. Collectively, these results indicated that BA may effectively inhibit pluripotency factor expression (Sox2, Oct4 and Nanog), EMT and the stem-like phenotype of pancreatic cancer cells via activating AMPK signaling. Therefore, BA may be considered an attractive therapeutic candidate and an effective inhibitor of the stem-like phenotype in pancreatic cancer cells. Further investigation into the development of BA as an anticancer drug is warranted.

e15170 Background: Tumor mutational burden (TMB) is an impressive predictive biomarker for immune checkpoint inhibitors therapy, and immunotherapy is one of the most promising methods for cancer treatment. However, the profile of TMB in various types of cancers was still poorly understood. Methods: In this study, genomic profiling of DNA was performed using next generation sequencing from a 539 genes panel in tumor tissues. The TMB was defined as the numbers of SNVs including synonymous and nonsynonymous mutations, and InDels per megabase in coding regions of sequenced genome. TMB-H was defined as highest mutation load quintile (top 20%) in each cancer type. The values of TMB of 874 patients were compared among eight main tumor types including 174 patients with liver cancer, 32 patients with bile duct cancer, 54 patients with gastric cancer, 119 patients with colorectal cancer, 27 patients with pancreatic cancer, 32 patients with melanoma, 25 patients with glioma and 411 patients with lung cancer cases by multiple independent samples nonparametric Kruskal-Wallis H test via SPSS v22.0. Results: The median values of TMB in liver cancer, bile duct cancer, gastric cancer, colorectal cancer, pancreatic cancer, melanoma, glioma and lung cancer were 10.29, 8.09, 11.03, 10.29, 5.88, 7.35, 5.88 and 7.35 Muts/Mb, respectively. The cut-off values of TMB-H in corresponding eight solid tumors were 16.18, 19.12, 19.85, 16.18, 10.29, 13.24, 10.29 and 18.38 Muts/Mb, respectively. Based on nonparametric Kruskal-Wallis H test, there were significant differences of median TMB values across eight independent tumor types (χ2 = 26.752, p = 3.693×10−4). Significant differences between patients with colorectal cancer and pancreatic cancer ( p = 0.005) or lung cancer ( p = 0.011) were observed via pairwise comparisons. Conclusions: Our study proved the presence of significant differences across eight cancer types, deepened the knowledge of the cancer-specific of TMB, provided useful information in immunology therapy for East Asian patients with solid tumors. As a retrospective study with a relatively small population, the conclusions of this study needed to be verified with a larger sample.

Simulating Clinical Features of Pancreatic Cancer in a Mouse Model with Specific Genetic Alterations in Kras and p53

Pancreatic cancer (PC) is highly lethal because of its immunosuppressive tumor microenvironment and resistance to immunotherapy. This study explored the role of NAT10-mediated N4-acetylcytidine (ac4C) RNA modification in pancreatic cancer progression and immune evasion. NAT10 (N-acetyltransferase 10) is overexpressed in pancreatic cancer tissues and correlates with poor prognosis. Mechanistically, NAT10 stabilizes ETS2 mRNA through ac4C acetylation, forming a positive feedback loop that upregulates NAT10 and PD-L1, thereby suppressing CD8 + T cell infiltration and promoting immune evasion. In addition, NAT10 stabilizes KRT8 mRNA via ac4C acetylation, which drives cancer cell proliferation and metastasis. Single-cell RNA sequencing analysis revealed enhanced interactions between pancreatic cancer epithelial cells with high NAT10 and KRT8 expression, and T cells, thereby providing new insights into the immune microenvironment. In vivo, NAT10 knockdown significantly inhibited tumor growth, enhanced CD8 + T cell infiltration, and reduced lung metastasis. Notably, combination therapy with an NAT10 inhibitor and anti-PD-L1 antibody demonstrated superior antitumor efficacy compared to monotherapy. In conclusion, NAT10 promotes pancreatic cancer progression and immune evasion by regulating the ETS2-PD-L1 axis and stabilizing KRT8 mRNA, highlighting its potential as a therapeutic target for overcoming immunotherapy resistance.