Sialyltransferase Inhibitors for the Treatment of Cancer Metastasis: Current Challenges and Future Perspectives.

Potent, cell-permeable, and subtype-selective sialyltransferase inhibitors represent an attractive family of substances that can potentially be used for the clinical treatment of cancer metastasis. These substances operate by specifically inhibiting sialyltransferase-mediated hypersialylation of cell surface glycoproteins or glycolipids, which then blocks the sialic acid recognition pathway and leads to deterioration of cell motility and invasion. A vast amount of evidence for the in vitro and in vivo effects of sialyltransferase inhibition or knockdown on tumor progression and tumor cell metastasis or colonization has been accumulated over the past decades. In this regard, this review comprehensively discusses the results of studies that have led to the recent discovery and development of sialyltransferase inhibitors, their potential biomedical applications in the treatment of cancer metastasis, and their current limitations and future opportunities.

Viewing Siglecs through the lens of tumor immunology.

Many Siglecs function as inhibitory receptors on innate and adaptive immune cells and may contribute to the attenuation of immune responses to tumors. Siglec 9 on neutrophils and Siglec 7 on NK cells are prominent examples of inhibitory Siglecs that can potentially dampen anti-tumor immunity. CD169 is a Siglec that may function as an adhesion molecule and a facilitator of the recognition and internalization of sialic acid decorated apoptotic bodies and exosomes derived from tumors. It can potentially contribute to both the attenuation as well as the facilitation of anti-tumor immunity. Siglecs have been best studied in the tumor context in animal models of cancer. Modulators of Siglec function are likely to be developed and investigated clinically in a cancer context over the next few years.

A Developed NK-92MI Cell Line with Siglec-7neg Phenotype Exhibits High and Sustainable Cytotoxicity against Leukemia Cells.

Altered sialic acid processing that leads to upregulation of cell surface sialylation is recognized as a key change in malignant tissue glycosylation. This cancer-associated hypersialylation directly impacts the signaling interactions between tumor cells and their surrounding microenvironment, especially the interactions mediated by immune cell surface sialic acid-binding immunoglobulin-like lectins (Siglecs) to relay inhibitory signals for cytotoxicity. First, we obtained a Siglec-7neg NK-92MI cell line, NK-92MI-S7N, by separating a group of Siglec-7neg cell population from an eight-month-long-term NK-92MI in vitro culture by fluorescence-activated cell sorting (FACS). The effect of Siglec-7 loss on NK-92MI-S7N cells was characterized by the cell morphology, proliferation, and cytotoxic activity via FACS, MTS assay, cytotoxic assay, and natural killer (NK) degranulation assay. We found the expression levels of Siglec-7 in NK-92MI were negatively correlated with NK cytotoxicity against leukemia cells. This NK-92MI-S7N cell not only shared very similar phenotypes with its parental cells but also possessed a high and sustainable killing activity. Furthermore, this Siglec-7neg NK line was unexpectedly capable of eliminating a NK-92MI-resistant leukemia cell, THP-1, through enhancing the effector-target interaction. In this study, a NK cell line with high and sustainable cytotoxicity was established and this cell may provide a potential application in NK-based treatment for leukemia patients.

Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer.

Certain pathogenic bacteria are known to modulate the innate immune response by decorating themselves with sialic acids, which can engage the myelomonocytic lineage inhibitory receptor Siglec-9, thereby evading immunosurveillance. We hypothesized that the well-known up-regulation of sialoglycoconjugates by tumors might similarly modulate interactions with innate immune cells. Supporting this hypothesis, Siglec-9-expressing myelomonocytic cells found in human tumor samples were accompanied by a strong up-regulation of Siglec-9 ligands. Blockade of Siglec-9 enhanced neutrophil activity against tumor cells in vitro. To investigate the function of inhibitory myelomonocytic Siglecs in vivo we studied mouse Siglec-E, the murine functional equivalent of Siglec-9. Siglec-E-deficient mice showed increased in vivo killing of tumor cells, and this effect was reversed by transgenic Siglec-9 expression in myelomonocytic cells. Siglec-E-deficient mice also showed enhanced immunosurveillance of autologous tumors. However, once tumors were established, they grew faster in Siglec-E-deficient mice. In keeping with this, Siglec-E-deficient macrophages showed a propensity toward a tumor-promoting M2 polarization, indicating a secondary role of CD33-related Siglecs in limiting cancer-promoting inflammation and tumor growth. Thus, we define a previously unidentified impact of inhibitory myelomonocytic Siglecs in cancer biology, with distinct roles that reflect the dual function of myelomonocytic cells in cancer progression. In keeping with this, a human polymorphism that reduced Siglec-9 binding to carcinomas was associated with improved early survival in non-small-cell lung cancer patients, which suggests that Siglec-9 might be therapeutically targeted within the right time frame and stage of disease.

Hypersialylation is a common feature of neurofibrillary tangles and granulovacuolar degenerations in Alzheimer's disease and tauopathy brains.

Glycosylation is one of the major post-translational modifications of proteins. The status of sialylation of the neuropathological hallmarks of various neurodegenerative disorders was investigated in this study. Here, we report the novel findings that two phosphorylated tau (p-tau)-containing structures associated with Alzheimer's disease (AD), that is, neurofibrillary tangles (NFTs) and granulovacuolar degenerations (GVDs), were hypersialylated. The NFTs, GVDs and dystrophic neurites of senile plaques (SPs) in AD hippocampi were clearly visualized by immunohistochemistry using an anti-sialic acid (SA) antibody. In contrast, the amyloid core of SPs was not sialylated at all. Interestingly, other p-tau-containing structures, that is, globose-type NFTs in progressive supranuclear palsy and Pick bodies and ballooned neurons in frontotemporal lobar degeneration with Pick bodies, were also hypersialylated. Unlike the p-tau-containing structures observed in tauopathies, the hallmarks of other neurodegenerative disorders, such as Lewy bodies in Parkinson's disease, glial cytoplasmic inclusions in multiple system atrophy, Bunina bodies, skein-like inclusions and round inclusions in amyotrophic lateral sclerosis, intranuclear inclusions in neuronal intranuclear inclusion disease and physiological bodies or granules (lipofuscin granules, corpora amylacea and melanin granules), were not immunolabeled by the anti-SA antibody. Because this antibody specifically identified NFTs and GVDs, immunostaining for sialylation represents a useful tool to screen these structures in a diagnostic setting. These results clearly indicate that the pathological hallmarks of various tauopathies are commonly hypersialylated, and that sialylation plays an important role in the process of p-tau accumulation in AD and other tauopathies.

Regulation of sialic acid metabolism in cancer.

Sialic acid, the terminal structure of cell surface glycans, has essential functions in regulating immune response, cell-to-cell communication, and cell adhesion. More importantly, an increased level of sialic acid, termed hypersialylation, has emerged as a commonly observed phenotype in cancer. Therefore, targeting sialic acid ligands (sialoglycans) and their receptors (Siglecs) may provide a new therapeutic approach for cancer immunotherapy. We highlight the complexity of the sialic acid metabolism and its involvement in malignant transformation within individual cancer subtypes. In this review, we focus on the dysregulation of sialylation, the intricate nature of sialic acid synthesis, and clinical perspective. We aim to provide a brief insight into the mechanism of hypersialylation and how our understanding of these processes can be leveraged for the development of novel therapeutics.

Recent advances in the development of sialyltransferase inhibitors to control cancer metastasis: A comprehensive review.

Metastasis accounts for the majority of cancer-associated mortalities, representing a huge health and economic burden. One of the mechanisms that enables metastasis is hypersialylation, characterized by an overabundance of sialylated glycans on the tumor surface, which leads to repulsion and detachment of cells from the original tumor. Once the tumor cells are mobilized, sialylated glycans hijack the natural killer T-cells through self-molecular mimicry and activatea downstream cascade of molecular events that result in inhibition of cytotoxicity and inflammatory responses against cancer cells, ultimately leading to immune evasion. Sialylation is mediated by a family of enzymes known as sialyltransferases (STs), which catalyse the transfer of sialic acid residue from the donor, CMP-sialic acid, onto the terminal end of an acceptor such as N-acetylgalactosamine on the cell-surface. Upregulation of STs increases tumor hypersialylation by up to 60% which is considered a distinctive hallmark of several types of cancers such as pancreatic, breast, and ovarian cancer. Therefore, inhibiting STs has emerged as a potential strategy to prevent metastasis. In this comprehensive review, we discuss the recent advances in designing novel sialyltransferase inhibitors using ligand-based drug design and high-throughput screening of natural and synthetic entities, emphasizing the most successful approaches. We analyse the limitations and challenges of designing selective, potent, and cell-permeable ST inhibitors that hindered further development of ST inhibitors into clinical trials. We conclude by analysing emerging opportunities, including advanced delivery methods which further increase the potential of these inhibitors to enrich the clinics with novel therapeutics to combat metastasis.

Aberrant Sialylation in Cancer: Therapeutic Opportunities.

The surface of every eukaryotic cell is coated in a thick layer of glycans that acts as a key interface with the extracellular environment. Cancer cells have a different 'glycan coat' to healthy cells and aberrant glycosylation is a universal feature of cancer cells linked to all of the cancer hallmarks. This means glycans hold huge potential for the development of new diagnostic and therapeutic strategies. One key change in tumour glycosylation is increased sialylation, both on N-glycans and O-glycans, which leads to a dense forest of sialylated structures covering the cell surface. This hypersialylation has far-reaching consequences for cancer cells, and sialylated glycans are fundamental in tumour growth, metastasis, immune evasion and drug resistance. The development of strategies to inhibit aberrant sialylation in cancer represents an important opportunity to develop new therapeutics. Here, I summarise recent advances to target aberrant sialylation in cancer, including the development of sialyltransferase inhibitors and strategies to inhibit Siglecs and Selectins, and discuss opportunities for the future.

Kolaviron Ameliorates 7, 12-Dimethylbenzanthracene - Induced Mammary Damage.

BACKGROUND: Kolaviron (KV) is a flavonoid-rich portion obtained from Garcinia kola seeds with a number of reported pharmacological effects. However, its ameliorative effects on 7,12-Dimethylbenzanthracene (DMBA)-induced mammary damage has not been fully investigated, despite the reported use of the seeds in the treatment of inflammatory related disorders. OBJECTIVE: To evaluate the ameliorative effects of KV on DMBA-induced mammary damage in female Wistar rats. METHODS: Forty-nine (49) female Wistar rats were randomly assigned into seven groups of seven rats each. DMBA was administered orally to rats in five of the groups as a single dose of 80 mg/kg body wt while the remaining two groups received the vehicle. The rats were palpated weekly for 3 months to monitor tumor formation. After 3 months of DMBA administration, 1 ml of blood was collected to assay for estrogen receptor- α (ER-α) level. Thereafter, the vehicle (dimethyl sulfoxide) was daily administered to the negative control and positive control groups for the 14 days duration of the experiment while three groups were each given a daily oral dose of 50, 100, and 200 mg/kg body wt of KV for the duration of the experiment. The last DMBA-induced group received 10 mg/kg body wt of the standard drug tamoxifen twice a week, and the remaining DMBA-free group received 200 mg/kg body wt KV. Subsequently, the animals were humanely sacrificed, and ER-α, sialic acids, sialidase, sialyltransferase levels were assayed in blood and mammary tissues followed by histopathological examinations. RESULTS: Significantly higher levels of estrogen receptor-α (ER-α), formation of lobular neoplastic cells, epithelial hyperplasia, lymphocyte infiltration, and increased sialylation were detected in DMBA-induced rats. Treatment with KV at 50, 100, and 200 mg/kg body weight resulted in a significant (p<0.05) decrease in ER-α level, free serum sialic acid (21.1%), the total sialic acid level of the mammary tissue (21.57%), sialyltransferase activity (30.83%) as well as mRNA level of the sialyltransferase gene (ST3Gal1) were observed after KV interventions. CONCLUSION: The findings suggest that KV could be further explored in targeting DMBA-induced mammary damage implicated in mammary carcinogenesis.

On-tissue spatially resolved glycoproteomics guided by N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape.

Here, we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of matrix-assisted laser desorption ionization (MALDI) N-glycan mass spectrometry imaging (MSI) and spatially resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, thus directly linking glycan imaging with intact glycopeptide identification. In total, our spatially resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slices and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.

Siglec-7 represents a glyco-immune checkpoint for non-exhausted effector memory CD8+ T cells with high functional and metabolic capacities.

While inhibitory Siglec receptors are known to regulate myeloid cells, less is known about their expression and function in lymphocytes subsets. Here we identified Siglec-7 as a glyco-immune checkpoint expressed on non-exhausted effector memory CD8+ T cells that exhibit high functional and metabolic capacities. Seahorse analysis revealed higher basal respiration and glycolysis levels of Siglec-7+ CD8+ T cells in steady state, and particularly upon activation. Siglec-7 polarization into the T cell immune synapse was dependent on sialoglycan interactions in trans and prevented actin polarization and effective T cell responses. Siglec-7 ligands were found to be expressed on both leukemic stem cells and acute myeloid leukemia (AML) cells suggesting the occurrence of glyco-immune checkpoints for Siglec-7+ CD8+ T cells, which were found in patients' peripheral blood and bone marrow. Our findings project Siglec-7 as a glyco-immune checkpoint and therapeutic target for T cell-driven disorders and cancer.

Tools to study and target the Siglec-sialic acid axis in cancer.

Siglecs are widely expressed on leucocytes and bind to ubiquitously presented glycans containing sialic acids (sialoglycans). Most Siglecs carry an immunoreceptor tyrosine-based inhibition motif (ITIM) and elicit an inhibitory intracellular signal upon ligand binding. A few Siglec receptors can, however, recruit immunoreceptor tyrosine-based activation motif (ITAM)-containing factors, which activate cells. The role of hypersialylation (the enhanced expression of sialoglycans) has recently been explored in cancer progression. Mechanistic studies have shown that hypersialylation on cancer cells can engage inhibitory Siglecs on the surface of immune cells and induce immunosuppression. These recent studies strongly suggest that the Siglec-sialic acid axis can act as a potential target for cancer immunotherapy. Moreover, the use of new tools and techniques is facilitating these studies. In this review, we summarise techniques used to study Siglecs, including different mouse models, monoclonal antibodies, Siglec fusion proteins, and sialoglycan arrays. Furthermore, we discuss the recent major developments in the study of Siglecs in cancer immunosuppression, tools, and techniques used in targeting the Siglec-sialic acid axis and the possibility of clinical intervention.

The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake.

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation-a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions.

Comprehensive Transcriptomic Analysis Identifies ST8SIA1 as a Survival-Related Sialyltransferase Gene in Breast Cancer.

Hypersialylation caused by the overexpression of sialyltransferases (STs) is a common feature in cancer that is associated with several characteristics of tumorigenesis. Thus, identifying cancer-associated STs is critical for cancer therapy. However, ST screening has been frequently conducted in cell line models. In this study, we conducted a comprehensive analysis of STs in the clinical database and identified the STs related with the survival of breast cancer patients. RNA sequencing (RNA-Seq) data of 496 patients were obtained from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA). Of the eight mapped STs, ST3GAL5, and ST8SIA1 met the acceptable area under the curve (AUC) criteria for overall survival (OS). Using Kaplan-Meier methods, we determined that high expression of ST8SIA1 was associated with poor 10-year OS in all patients, triple-negative breast cancer (TNBC), and non-TNBC patients, and poor disease-free survival (DFS) rates particularly in TNBC. ST8SIA1 also had superior AUC values in terms of OS/DFS. High ST8SIA1 levels showed a higher risk for poor OS in different groups of patients and a higher risk for poor DFS particularly in TNBC. In summary, we conducted a comprehensive analysis of STs from the clinical database and identified ST8SIA1 as a crucial survival-related ST, which might be a potential therapeutic target for breast cancer and TNBC patients.

Density functional theory based probe of the affinity interaction of saccharide ligands with extra-cellular sialic acid residues.

Changes in glycosylation pattern leads to malignant transformations among the cells. In combination with upregulated actions of sialyltransferases, it ultimately leads to differential expression of sialic acid (SA) at cell surface. Given its negative charge and localization to extracellular domain, SA has been exploited for the development of targeted theranostics using approaches, such as, cationization and appending recognition saccharides on carrier surface. In this study, we have performed quantum mechanical calculations based on density functional theory (DFT) to study the interaction of saccharides with extracellular SA. Gradient-corrected DFT with the three parameter function (B3) was utilized for the calculation of Lee-Yang-Parr (LYP) correlation function. Atomic charge, vibrational frequencies and energy of the optimized structures were calculated through B3LYP. Our calculations demonstrate a stronger galactose-sialic acid interaction at tumour-relevant low pH and hyperthermic condition. These results support the application of pH responsive delivery vehicles and targeted hyperthermic chemotherapy for eradicating solid tumour deposits. These studies, conducted a priori, can guide the formulation scientists over appropriate choice of ligands and their applications in the design of 'smart' theranostic tools.

Application of surface enhanced Raman spectroscopy as a diagnostic system for hypersialylated metastatic cancers.

Early diagnosis of metastatic cancers could greatly limit the number of cancer-associated deaths. Aberrant surface expression of sialic acid (hypersialylation) on tumors correlating with metastatic incidence and its involvement in tumorigenesis and progression is widely reported; hence detection of hypersialylated tumors may be an effective strategy to identify metastatic cancers. We herein report on the application of phenylboronic acid-installed PEGylated gold nanoparticles coupled with Toluidine blue O (T/BA-GNPs) as SERS probes to target surface sialic acid (N-acetylneuraminic acid, Neu5Ac). Strong SERS signals from metastatic cancer cell lines (breast cancer; MDA-MB231 and colon cancer; Colon-26) were observed, contrary to non-metastatic MCF-7 cells (breast cancer). The detected SERS signals from various cancer cell lines correlated with their reported metastatic potential, implying that our T/BA-GNP based SERS system was capable of distinguishing the metastaticity of cells based on the surface Neu5Ac density. T/BA-GNP based SERS system could also significantly differentiate between hypersialylated tumor tissues and healthy tissues with high SERS signal to noise ratio, due to plasmon coupling between the specifically aggregated functionalized GNPs. Furthermore, we also confirmed reduction in SERS signals from MDA-MB231 surface upon treatment with our original reactive oxygen species (ROS)-scavenging polymeric micelle, nitroxide-radical containing nanoparticles (RNPs). The ROS-mediated abrogation of sialylation by impairing the activation of NF-κB-sialyltransferase signaling cascade upon RNP treatment was confirmed by expression studies and the T/BA-GNPs based SERS system. The aforementioned findings thus, establish T/BA-GNPs based SERS as a potential cytodiagnostic system to detect hypersialylated metastatic tumors and RNPs as anti-metastatic cancer drug candidates.