Neisserial adhesin A (NadA) binds human Siglec-5 and Siglec-14 with high affinity and promotes bacterial adhesion/invasion.

Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the KD value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death.

MicroRNA:Siglec crosstalk in cancer progression.

Aberrant Siglec expression in the tumour microenvironment has been implicated in tumour malignancies and can impact tumour behaviour and patient survival. Further to this, engagement with sialoglycans induces masked antigen recognition and promotes immune evasion, highlighting deregulated immune function. This necessitates the elucidation of their expression profiles in tumour progression. MicroRNAs (miRNAs) mediated targeting represents a novel approach to further elucidate Siglec potential and clinical relevance. Although miRNA activity in Siglec expression remains limited, we highlight current literature detailing miRNA:Siglec interactions within the tumour landscape and provide insights for possible diagnostic and therapeutic strategies in targeting the Siglec/sialic acid axis.

Dissecting the abilities of murine Siglecs to interact with gangliosides.

Siglecs are cell surface receptors whose functions are tied to the binding of their sialoglycan ligands. Recently, we developed an optimized liposome formulation and used it to investigate the binding of human Siglecs (hSiglec) against a panel of gangliosides. Animal models, more specifically murine models, are used to understand human biology; however, species-specific differences can complicate the interpretation of the results. Herein, we used our optimized liposome formulation to dissect the interactions between murine Siglecs (mSiglecs) and gangliosides to assess the appropriateness of mSiglecs as a proxy to better understand the biological roles of hSiglec-ganglioside interactions. Using our optimized liposome formulation, we found that ganglioside binding is generally conserved between mice and humans with mSiglec-1, -E, -F, and -15 binding multiple gangliosides like their human counterparts. However, in contrast to the hSiglecs, we observed little to no binding between the mSiglecs and ganglioside GM1a. Detailed analysis of mSiglec-1 interacting with GM1a and its structural isomer, GM1b, suggests that mSiglec-1 preferentially binds α2-3-linked sialic acids presented from the terminal galactose residue. The ability of mSiglecs to interact or not interact with gangliosides, particularly GM1a, has implications for using mice to study neurodegenerative diseases, infections, and cancer, where interactions between Siglecs and glycolipids have been proposed to modulate these human diseases.

Sialic Acid-Siglec-E Interactions Regulate the Response of Neonatal Macrophages to Group B Streptococcus.

The mammalian Siglec receptor sialoadhesin (Siglec1, CD169) confers innate immunity against the encapsulated pathogen group B Streptococcus (GBS). Newborn lung macrophages have lower expression levels of sialoadhesin at birth compared with the postnatal period, increasing their susceptibility to GBS infection. In this study, we investigate the mechanisms regulating sialoadhesin expression in the newborn mouse lung. In both neonatal and adult mice, GBS lung infection reduced Siglec1 expression, potentially delaying acquisition of immunity in neonates. Suppression of Siglec1 expression required interactions between sialic acid on the GBS capsule and the inhibitory host receptor Siglec-E. The Siglec1 gene contains multiple STAT binding motifs, which could regulate expression of sialoadhesin downstream of innate immune signals. Although GBS infection reduced STAT1 expression in the lungs of wild-type newborn mice, we observed increased numbers of STAT1+ cells in Siglece-/- lungs. To test if innate immune activation could increase sialoadhesin at birth, we first demonstrated that treatment of neonatal lung macrophages ex vivo with inflammatory activators increased sialoadhesin expression. However, overcoming the low sialoadhesin expression at birth using in vivo prenatal exposures or treatments with inflammatory stimuli were not successful. The suppression of sialoadhesin expression by GBS-Siglec-E engagement may therefore contribute to disease pathogenesis in newborns and represent a challenging but potentially appealing therapeutic opportunity to augment immunity at birth.

Identification of therapeutic targets and prognostic biomarkers in the Siglec family of genes in tumor immune microenvironment of sarcoma.

Sarcomas (SARC) are a highly heterogeneous cancer type that is prone to recurrence and metastasis. Numerous studies have confirmed that Siglecs are involved in immune signaling and play a key role in regulating immune responses in inflammatory diseases and various cancers. However, studies that systematically explore the therapeutic and prognostic value of Siglecs in SARC patients are very limited. The online databases GEPIA, UALCAN, TIMER, The Kaplan-Meier Plotter, GeneMANIA, cBioPortal, and STING were used in this study. IHC staining was performed on the collected patient tissues, and clinical data were statistically analyzed. The transcript levels of most Siglec family members showed a high expression pattern in SARC. Compared with normal tissues, Siglec-5, Siglec-10, and Siglec-12 were abnormally highly expressed in tumor tissues. Importantly, Siglec-15 was significantly associated with poor prognosis. Functional enrichment analysis showed that the Siglec family was mainly enriched in hematopoietic cell lineages. The genes associated with molecular mutations in the Siglec family were mainly TP53 and MUC16, among which Siglec-2 and Siglec-15 were significantly associated with the survival of patients. The expression levels of all Siglec family members were significantly correlated with various types of immune cells (B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils and dendritic cells). Furthermore, a significant correlation was found between the somatic copy number changes of all Siglec molecules and the abundance of immune infiltrates. Our study paints a promising vision for the development of immunotherapy drugs and the construction of prognostic stratification models by investigating the therapeutic and prognostic potential of the Siglec family for SARC.

Targeting myeloid cells for cancer immunotherapy: Siglec-7/9/10/15 and their ligands.

Advances in immunotherapy have revolutionized cancer treatment, yet many patients do not show clinical responses. While most immunotherapies target T cells, myeloid cells are the most abundant cell type in solid tumors and are key orchestrators of the immunosuppressive tumor microenvironment (TME), hampering effective T cell responses. Therefore, unraveling the immune suppressive pathways within myeloid cells could unveil new avenues for cancer immunotherapy. Over the past decade, Siglec receptors and their ligand, sialic acids, have emerged as a novel immune checkpoint on myeloid cells. In this review, we highlight key findings on how sialic acids modify immunity in the TME through engagement of Siglec-7/9/10/15 expressed on myeloid cells, and how the sialic acid-Siglec axis can be targeted for future cancer immunotherapies.

Genome-wide determinants of cellular immune responses to mumps vaccine.

BACKGROUND: We have previously described genetic polymorphisms in candidate genes that are associated with inter-individual variations in antibody responses to mumps vaccination. To expand upon our previous work, we performed a genome-wide association study (GWAS) to discover host genetic variants associated with mumps vaccine-induced cellular immune responses. METHODS: We performed a GWAS of mumps-specific immune response outcomes (11 secreted cytokines/chemokines) in a cohort of 1,406 subjects. RESULTS: Among the 11 cytokine/chemokines we studied, four (IFN-γ, IL-2, IL-1ß, and TNFα) demonstrated GWAS signals reaching genome-wide significance (p < 5 × 10-8). A genomic region (encoding Sialic acid-binding immunoglobulin-type lectins/SIGLEC) located on chromosome 19q13 (p < 5 × 10-8) was associated with both IL-1ß and TNFα responses. The SIGLEC5/SIGLEC14 region contained 11 statistically significant single nucleotide polymorphisms (SNPs), including the intronic SIGLEC5 rs872629 (p = 1.3E-11) and rs1106476 (p = 1.32E-11) whose alternate alleles were significantly associated with decreased levels of mumps-specific IL-1ß (rs872629, p = 1.77E-09; rs1106476, p = 1.78E-09) and TNFα (rs872629, p = 1.3E-11; rs1106476, p = 1.32E-11) production. CONCLUSIONS: Our results suggest that SNPs in the SIGLEC5/SIGLEC14 genes play a role in cellular and inflammatory immune responses to mumps vaccination. These findings motivate further research into the functional roles of SIGLEC genes in the regulation of mumps vaccine-induced immunity.

The inhibitory receptor Siglec-G controls the severity of chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eµ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.

Multi-omics analysis of Siglec family genes in cutaneous melanoma.

Background: Melanoma is widely recognized as the most aggressive and fatal type of skin cancer; however, effective prognostic markers are lacking. The sialic acid-binding immunoglobulin-type lectin (Siglec) gene family plays an important role in the development of tumors and immune escape, but its prognostic role in melanoma remains unknown. Results: Siglec genes have a high mutation frequency, with up to 8% in SIGLEC7. High expression levels of Siglecs in tumor bulk suggests a better prognosis. Siglecs also show a high degree of synergistic expression. Immunohistochemistry was used to analyze the expression of SIGLEC9 in tumor tissue microarray. The expression of SIGLEC9 in tumor tissue without metastasis was higher than that in tumor tissue with metastasis. We used unsupervised clustering to create a high expression of Siglec (HES) cluster and a low expression of Siglec (LES) cluster. The HES cluster correlated with high overall survival and increased expression levels of Siglec genes. The HES cluster also showed significant immune cell infiltration and activation of immune signaling pathways. We used least absolute shrinkage and selection operator (LASSO) regression analysis to reduce the dimensionality of Siglec cluster-related genes and constructed a prognostic model composed of SRGN and GBP4, which can risk-stratify patients in both the training and test datasets. Conclusion: We conducted a multi-omics analysis of the Siglec family genes in melanoma and found that Siglecs play an important role in the occurrence and development of melanoma. Typing constructed using Siglecs can show risk stratification and derived prognostic models can predict a patient's risk score. In summary, Siglec family genes are potential targets for melanoma treatment as well as prognostic markers that can direct individualized treatments and improve overall survival.

Histone deacetylation-regulated cell surface Siglec-7 expression promoted megakaryocytic maturation and enhanced platelet-like particle release.

BACKGROUND: Functioning as important hematologic cells for hemostasis, wound healing and immune defense platelets are produced before being released into the blood by cytoplasmic fragmentation at the end of the megakaryocyte (MK) differentiation, during which the involvement of both apoptosis and autophagy has been reported. Inhibitory sialic acid-binding immunoglobulin-like lectin-7 gene (Siglec-7) can be expressed on platelets and induce apoptosis on activation for uncharacterized function. OBJECTIVE: We aimed to investigate the regulatory mechanism for Siglec-7 activation along MK differentiation and its physiologic role during the MK maturation and platelet formation. METHODS: By using 2 well-established MK differentiation models (HEL and K562) and human primary CD34+ cell, we examined the upregulations of transcript and protein levels of Siglec-7 during MK differentiation, and the effect of Siglec-7 surface presence on MK differentiation and platelet-like particles (PLPs) release. RESULTS: We show that both transcripts and surface Siglec-7 were elevated during MK differentiation, and the histone deacetylase 1 (HDAC1) acted as a negative regulator for Siglec-7 activation. By increasing Siglec-7 surface expression, we found that increased presence of Siglec-7 not only enhanced MK maturation but also the release of PLPs by activating caspase 3-dependent signaling, as evidenced in the observation of more CD41, polyploidy, and platelet factor 4 transcript formations. CONCLUSION: In this study, we demonstrated that Siglec-7 activation was subjected to epigenetic regulation, and the resulting induced expression of surface Siglec-7 played an important regulatory role in promoting MK differentiation, maturation, and PLP formation.

Siglec receptors as new immune checkpoints in cancer.

Cancer immunotherapy in the form of immune checkpoint inhibitors and cellular therapies has improved the treatment and prognosis of many patients. Nevertheless, most cancers are still resistant to currently approved cancer immunotherapies. New approaches and rational combinations are needed to overcome these resistances. There is emerging evidence that Siglec receptors could be regarded as new immune checkpoints and targets for cancer immunotherapy. In this review, we summarize the experimental evidence supporting Siglec receptors as new immune checkpoints in cancer and discuss their mechanisms of action, as well as current efforts to target Siglec receptors and their interactions with sialoglycan Siglec-ligands.

Human sialoglycan ligands for immune inhibitory Siglecs.

Most human Siglecs (sialic acid binding immunoglobulin-like lectins) are expressed on the surfaces of overlapping subsets of immune cells, and most carry immunoreceptor tyrosine-based inhibitory domains on their intracellular motifs. When immune inhibitory Siglecs bind to complementary sialoglycans in their local milieu, engagement results in down-regulation of the immune response. Siglecs have come under scrutiny as potential targets of drugs to modify the course of inflammation (and other immune system responses) and as immune checkpoints in cancer. Human Siglecs bind to endogenous human sialoglycans. The identities of these endogenous human sialoglycan immune regulators are beginning to emerge, along with some general principles that may inform future investigations in this area. Among these principles is the finding that a cell type or tissue may express a ligand for a particular Siglec on a single or a very few of its sialoglycoproteins. The selected protein carrier for a particular Siglec may be unique in a certain tissue, but vary tissue-to-tissue. The binding affinity of endogenous Siglec ligands may surpass that of its binding to synthetic sialoglycan determinants by several orders of magnitude. Since most human Siglecs have evolved rapidly and are distinct from those in most other mammals, this review describes endogenous human Siglec ligands for several human immune inhibitory Siglecs. As the identities of these immune regulatory sialoglycan ligands are defined, additional opportunities to target Siglecs therapeutically may emerge.

Discovery, classification, evolution and diversity of Siglecs.

Immunoglobulin (Ig) superfamily proteins play diverse roles in vertebrates, including regulation of cellular responses by sensing endogenous or exogenous ligands. Siglecs are a family of glycan-recognizing proteins belonging to the Ig superfamily (i.e., I-type lectins). Siglecs are expressed on various leukocyte types and are involved in diverse aspects of immunity, including the regulation of inflammatory responses, leukocyte proliferation, host-microbe interaction, and cancer immunity. Sialoadhesin/Siglec-1, CD22/Siglec-2, and myelin-associated glycoprotein/Siglec-4 were among the first to be characterized as members of the Siglec family, and along with Siglec-15, they are relatively well-conserved among tetrapods. Conversely, CD33/Siglec-3-related Siglecs (CD33rSiglecs, so named as they show high sequence similarity with CD33/Siglec-3) are encoded in a gene cluster with many interspecies variations and even intraspecies variations within some lineages such as humans. The rapid evolution of CD33rSiglecs expressed on leukocytes involved in innate immunity likely reflects the selective pressure by pathogens that interact and possibly exploit these Siglecs. Human Siglecs have several additional unique and/or polymorphic properties as compared with closely related great apes, changes possibly related to the loss of the sialic acid Neu5Gc, another distinctly human event in sialobiology. Multiple changes in human CD33rSiglecs compared to great apes include many examples of human-specific expression in non-immune cells, coinciding with human-specific diseases involving such cell types. Some Siglec gene polymorphisms have dual consequences-beneficial in a situation but detrimental in another. The association of human Siglec gene polymorphisms with several infectious and non-infectious diseases likely reflects the ongoing competition between the host and microbial pathogens.

Expression and function of Siglec-15 in RLPS and its correlation with PD-L1: Bioinformatics Analysis and Clinicopathological Evidence.

Purpose: Retroperitoneal liposarcoma (RLPS) is a rare malignancy without effective treatment. Since current treatment for unresectable RLPS is unsatisfactory, immunotherapy and targeted therapy are urgently needed. Siglec-15 is a transmembrane protein highly homologous to PD-L1 and is involved in tumor immune escape. The biological function of Siglec-15 in RLPS, its prognostic relevance and its relationship with PD-L1 need to be further clarified. In this study, we aimed to explore the biological function of Siglec-15 in sarcomas through bioinformatics analysis, and we also evaluated Siglec-15 and PD-L1 expression in RLPS samples. The relationship between the expression of Siglec-15 and PD-L1 and their clinicopathological relevance and prognostic value were also investigated in clinical RLPS patients. Methods: The RNA sequencing data of 259 sarcoma cases and 48 RLPS cases from TCGA were used to analyze the Siglec-15 expression and the differentially expressed genes (DEG) related with Siglec-15 expression. In addition, DEGs were subsequently analyzed through the gene ontology (GO)/ Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network. Tumor specimens were obtained from 91 RLPS patients of our sarcoma center, and Siglec-15 and PD-L1 expression were evaluated using immunohistochemistry. The correlation between the expression level of these two markers as well as their correlation with clinicopathological factors and prognosis of RLPS patients was also assessed. Results: GEPIA analysis showed that the high expression of Siglec-15 was associated with poor sarcoma OS (P=0.034). A total of 682 differential genes were identified between the high and low expression groups of Siglec-15 in RLPS. Enrichment analysis of the KEGG pathway showed that Siglec-15 was related to the Hippo signaling pathway and the neuroactive ligand-receptor interaction. GO annotation analysis showed that the expression of Siglec-15 may thus be able to affect serine hydrolase activity, alongside signal receptor activator activity. The top 5 genes with the largest number of connection points are APOA1, F2, AHSG, AMBP, SERPINC1. In subsequent studies, we used 91 liposarcoma samples from our center for verification. Siglec-15 was expressed in 84.6% of RLPS cases, whereas PD-L1 was expressed in 17.6% of RLPS cases. A negative correlation was observed between Siglec-15 and PD-L1 expression (P=0.020). In this group of RLPS patients, high Siglec-15 expression was correlated with poorer disease-free survival (DFS) (P=0.021), and it was an independent predictor of DFS (hazard ratio: 2.298; 95% confidence interval: 1.154-4.576; P=0.018). However, we did not find a correlation between PD-L1 expression and overall survival or DFS in RLPS patients. Conclusion: The DEG and signaling pathways identified in the study could provide a preliminary understanding of the underlying molecular mechanisms of Siglec-15 in the development and progression of RLPS. High expression of Siglec-15 was a negative independent predictive factor for DFS of RLPS. The negative relationship between Siglec-15 and PD-L1 expression suggested that the Siglec-15 pathway might be an important supplement to PD-L1 treatment.

Targeting cancer glycosylation repolarizes tumor-associated macrophages allowing effective immune checkpoint blockade.

Immune checkpoint blockade (ICB) has substantially improved the prognosis of patients with cancer, but the majority experiences limited benefit, supporting the need for new therapeutic approaches. Up-regulation of sialic acid-containing glycans, termed hypersialylation, is a common feature of cancer-associated glycosylation, driving disease progression and immune escape through the engagement of Siglec receptors on tumor-infiltrating immune cells. Here, we show that tumor sialylation correlates with distinct immune states and reduced survival in human cancers. The targeted removal of Siglec ligands in the tumor microenvironment, using an antibody-sialidase conjugate, enhanced antitumor immunity and halted tumor progression in several murine models. Using single-cell RNA sequencing, we revealed that desialylation repolarized tumor-associated macrophages (TAMs). We also identified Siglec-E as the main receptor for hypersialylation on TAMs. Last, we found that genetic and therapeutic desialylation, as well as loss of Siglec-E, enhanced the efficacy of ICB. Thus, therapeutic desialylation represents an immunotherapeutic approach to reshape macrophage phenotypes and augment the adaptive antitumor immune response.

Landscape of exitrons in gastric cancer.

BACKGROUND: Exitron is a new type of non-canonical alternative splicing. Accumulating evidence implies exitron may have pathological function and contribute to another source of anti-tumor immunogenicity in various cancers. Its role in gastric cancer remains poorly understood. Large-scale, multi-omics analysis could comprehensively characterize the landscape of exitrons in gastric cancer, reveal undiscovered mechanism and hopefully identify molecular biomarkers for predicting immunotherapy response. METHODS: We collected datasets from five studies for analysis. RNA sequencing was used for exitron identification. Somatic mutations were detected by whole exome sequencing. Neopeptides were confirmed by proteome mass spectrometry. FINDINGS: 42174 gastric cancer-specific exitrons (GCSEs) were identified in 632 patients. GCSEs were clinically relevant to gender, age, Lauren type, tumor stage and prognosis. Tissue specificity test and pathogenic exitron prediction revealed their unique functional impact. GCSEs were mutually exclusive with mutations and demonstrated both unique and complementary function against TP53 mutation in gastric cancer. We further established splicing regulatory network to reveal upstream regulation of exitron splicing. We also evaluated the immunogenicity and diagnostic potential of GCSEs. Evidence of GCSEs-derived neopeptide expression was validated by whole proteome mass spectrometry. PD-1 and Siglecs were significantly increased in high neoantigen load patients. But exitron-related biomarkers failed to predict immunotherapy response, possibly due to small sample size and insufficient sequencing depth. INTERPRETATION: The present study provided a comprehensive multidimensional landscape of gastric cancer exitrons and underscores insights into underexplored mechanism in gastric cancer pathology. FUNDING: The Guangdong Provincial Key Laboratory of Precision Medicine for Gastroinstestinal Cancer (2020B121201004), the Guangdong Provincial Major Talents Project (No. 2019JC05Y361) and National Natural Science Foundation of China (grant number:82172960 and 81872013).

Identification and validation of a siglec-based and aging-related 9-gene signature for predicting prognosis in acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) is a group of highly heterogenous and aggressive blood cancer. Despite recent progress in its diagnosis and treatment, patient outcome is variable and drug resistance results in increased mortality. The siglec family plays an important role in tumorigenesis and aging. Increasing age is a risk factor for AML and cellular aging contributes to leukemogenesis via various pathways. METHODS: The differential expression of the siglec family was compared between 151 AML patients and 70 healthy controls, with their information downloaded from TCGA and GTEx databases, respectively. How siglec expression correlated to AML patient clinical features, immune cell infiltration, drug resistance and survival outcome was analyzed. Differentially expressed genes in AML patients with low- and high-expressed siglec9 and siglec14 were analyzed and functionally enriched. The aging-related gene set was merged with the differentially expressed genes in AML patients with low and high expression of siglec9, and merged genes were subjected to lasso regression analysis to construct a novel siglec-based and aging-related prognostic model. The prediction model was validated using a validation cohort from GEO database (GSE106291). RESULTS: The expression levels of all siglec members were significantly altered in AML. The expression of siglecs was significantly correlated with AML patient clinical features, immune cell infiltration, drug resistance, and survival outcome. Based on the differentially expressed genes and aging-related gene set, we developed a 9-gene prognostic model and decision curve analysis revealed the net benefit generated by our prediction model. The siglec-based and aging-related 9-gene prognostic model was tested using a validation data set, in which AML patients with higher risk scores had significantly reduced survival probability. Time-dependent receiver operating characteristic curve and nomogram were plotted and showed the diagnostic accuracy and predictive value of our 9-gene prognostic model, respectively. CONCLUSIONS: Overall, our study indicates the important role of siglec family in AML and the good performance of our novel siglec-based and aging-related 9-gene signature in predicting AML patient outcome.

[Study on the effect and mechanisms of SIGLEC-15 on laryngeal squamous cell carcinoma].

Objective: To investigate the effect of sialic acid combined with immunoglobulin-like lectin 15 (SIGLEC-15) on laryngeal squamous cell carcinoma (LSCC) and underlying mechanisms. Methods: The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Profiling Interactive Analysis (GEPIA2) databases were used for bioinformatics analysis. Cell Counting Kit-8 (CCK8), flow cytometry, and Transwell method were used respectively to detect proliferation, apoptosis, cell cycle, metastasis and invasion behaviors of the cells. Gene chip method was used for detecting up-regulated and down-regulated genes and performing enrichment analysis of differential genes. Western Blotting (WB) and Quantitative Real-time PCR (qPCR) were used to detect the expressions of proteins. Tumor formation experiments in nude mice were used to detect the effect of SIGLEC-15 on the growth of transplanted tumors. Wilcoxon rank sum test, t-test and Log-rank test were used for statistical analysis. Results: SIGLEC-15 was highly expressed in laryngeal squamous cell carcinoma and closely related to life in being. TU686SIGLEC-15-with low expression of SIGLEC-15 was constructed. Compared to TU686SIGLEC-15+, TU686SIGLEC-15-showed significantly reduced activities of proliferation (48 h: 1.32±0.23 vs. 2.56±0.37, t=6.59, P<0.05), migration (1 036.52±51.22 vs. 1 819.62±180.24, t=7.22, P<0.05) and invasion (469.21±112.25 vs. 961.45±102.03, t=7.85, P<0.05); early increased apoptosis ((23.27±1.12)% vs. (5.64±1.61)%, t=11.32, P<0.05); blocked cell cycle at G0/G1 ((59.32±3.65)% vs. (35.46±3.57)%, t=9.85, P<0.05); the knockdown of SIGLEC-15 resulted in up-regulation of 864 genes, down-regulation of 357 genes, with significant changes in molecules of cell cycle, apoptosis and JAK/STAT signal pathways, and the expressions of p-JAK2, p-STAT3, Caspase-3, Bad, Bcl-2, and Cyclin d1 proteins. Tumor formation experiments in nude mice showed that at 8 weeks after the tumors were implanted, the growth transplanted tumors of TU686 SIGLEC-15-cell group was slower than that of TU686 SIGLEC-15+cell group, with significant difference in the mean tumor weights between two groups ((0.382±0.054) g vs. (1.277±0.126) g, t=8.44, P<0.05), while the expression of SIGLEC-15 was lower in the transplanted tumors of SIGLEC-15 knockdown group compared to control group, with significant difference(11.29±2.17 vs. 36.25±7.56, t=9.28, P<0.05). Conclusion: SIGLEC-15 is highly expressed in LSCC and can promote the progression of LSCC through the JAK2-STAT3 pathway.

Molecular Basis and Role of Siglec-7 Ligand Expression on Chronic Lymphocytic Leukemia B Cells.

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is an immune checkpoint-like glycan recognition protein on natural killer (NK) cells. Cancer cells often upregulate Siglec ligands to subvert immunosurveillance, but the molecular basis of Siglec ligands has been elusive. In this study, we investigated Siglec-7 ligands on chronic lymphocytic leukemia (CLL) B cells. CLL B cells express higher levels of Siglec-7 ligands compared with healthy donor B cells, and enzymatic removal of sialic acids or sialomucins makes them more sensitive to NK cell cytotoxicity. Gene knockout experiments have revealed that the sialyltransferase ST6GalNAc-IV is responsible for the biosynthesis of disialyl-T (Neu5Acα2-3Galß1-3[Neu5Acα2-6]GalNAcα1-), which is the glycotope recognized by Siglec-7, and that CD162 and CD45 are the major carriers of this glycotope on CLL B cells. Analysis of public transcriptomic datasets indicated that the low expression of GCNT1 (encoding core 2 GlcNAc transferase, an enzyme that competes against ST6GalNAc-IV) and high expression of ST6GALNAC4 (encoding ST6GalNAc-IV) in CLL B cells, together enhancing the expression of the disialyl-T glycotope, are associated with poor patient prognosis. Taken together, our results determined the molecular basis of Siglec-7 ligand overexpression that protects CLL B cells from NK cell cytotoxicity and identified disialyl-T as a potential prognostic marker of CLL.

Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells.

Mast cells are tissue-resident cells that contribute to allergic diseases, among others, due to excessive or inappropriate cellular activation and degranulation. Therapeutic approaches to modulate mast cell activation are urgently needed. Siglec-6 is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptor selectively expressed by mast cells, making it a promising target for therapeutic intervention. However, the effects of its engagement on mast cells are poorly defined. Siglec-6 expression and endocytosis on primary human mast cells and mast cell lines were assessed by flow cytometry. SIGLEC6 mRNA expression was examined by single-cell RNAseq in esophageal tissue biopsy samples. The ability of Siglec-6 engagement or co-engagement to prevent primary mast cell activation was determined based on assessments of mediator and cytokine secretion and degranulation markers. Siglec-6 was highly expressed by all mast cells examined, and the SIGLEC6 transcript was restricted to mast cells in esophageal biopsy samples. Siglec-6 endocytosis occurred with delayed kinetics relative to the related receptor Siglec-8. Co-crosslinking of Siglec-6 with FcεRIα enhanced the inhibition of mast cell activation and diminished downstream ERK1/2 and p38 phosphorylation. The selective, stable expression and potent inhibitory capacity of Siglec-6 on human mast cells are favorable for its use as a therapeutic target in mast cell-driven diseases.