The pro-inflammatory cytokines IL-1ß and IL-6 promote upregulation of the ST6GAL1 sialyltransferase in pancreatic cancer cells.

The ST6GAL1 sialyltransferase is overexpressed in multiple cancers, including pancreatic ductal adenocarcinoma (PDAC). ST6GAL1 adds an α2-6-linked sialic acid to N-glycosylated membrane receptors, which consequently modulates receptor structure and function. While many studies have investigated the effects of ST6GAL1 on cell phenotype, there is a dearth of knowledge regarding mechanisms that regulate ST6GAL1 expression. In the current study, we evaluated the regulation of ST6GAL1 by two pro-inflammatory cytokines, IL-1ß and IL-6, which are abundant within the PDAC tumor microenvironment. Cytokine activity was monitored using the Suit-2 PDAC cell line and two Suit-2-derived metastatic subclones, S2-013 and S2-LM7AA. For all three cell models, treatment with IL-1ß or IL-6 increased the expression of ST6GAL1 protein and mRNA. Specifically, IL-1ß and IL-6 induced expression of the ST6GAL1 YZ mRNA isoform, which is driven by the P3 promoter. The ST6GAL1 H and X isoforms were not detected. Promoter reporter assays confirmed that IL-1ß and IL-6 activated transcription from the P3 promoter. We then examined downstream signaling mechanisms. IL-1ß is known to signal through the NFκB transcription factor, whereas IL-6 signals through the STAT3 transcription factor. CUT&RUN experiments revealed that IL-1ß promoted the binding of NFκB to the ST6GAL1 P3 promoter, and IL-6 induced the binding of STAT3 to the P3 promoter. Finally, we determined that inhibitors of NFκB and STAT3 blocked the upregulation of ST6GAL1 stimulated by IL-1ß and IL-6, respectively. Together, these results highlight a novel molecular pathway by which cytokines within the tumor microenvironment stimulate the upregulation of ST6GAL1 in PDAC cells.

BET inhibition decreases HMGCS2 and sensitizes resistant pancreatic tumors to gemcitabine.

Efforts to develop targetable molecular bases for drug resistance for pancreatic ductal adenocarcinoma (PDAC) have been equivocally successful. Using RNA-seq and ingenuity pathway analysis we identified that the superpathway of cholesterol biosynthesis is upregulated in gemcitabine resistant (gemR) tumors using a unique PDAC PDX model with resistance to gemcitabine acquired in vivo. Analysis of additional in vitro and in vivo gemR PDAC models showed that HMG-CoA synthase 2 (HMGCS2), an enzyme involved in cholesterol biosynthesis and rate limiting in ketogenesis, is overexpressed in these models. Mechanistic data demonstrate the novel findings that HMGCS2 contributes to gemR and confers metastatic properties in PDAC models, and that HMGCS2 is BRD4 dependent. Further, BET inhibitor JQ1 decreases levels of HMGCS2, sensitizes PDAC cells to gemcitabine, and a combination of gemcitabine and JQ1 induced regressions of gemR tumors in vivo. Our data suggest that decreasing HMGCS2 may reverse gemR, and that HMGCS2 represents a useful therapeutic target for treating gemcitabine resistant PDAC.

ST6GAL1 is associated with poor response to chemoradiation in rectal cancer.

INTRODUCTION: Colorectal cancer is the third most common cause of cancer death. Rectal cancer makes up a third of all colorectal cases. Treatment for locally advanced rectal cancer includes chemoradiation followed by surgery. We have previously identified ST6GAL1 as a cause of resistance to chemoradiation in vitro and hypothesized that it would be correlated with poor response in human derived models and human tissues. METHODS: Five organoid models were created from primary human rectal cancers and ST6GAL1 was knocked down via lentivirus transduction in one model. ST6GAL1 and Cleaved Caspase-3 (CC3) were assessed after chemoradiation via immunostaining. A tissue microarray (TMA) was created from twenty-six patients who underwent chemoradiation and had pre- and post-treatment specimens of rectal adenocarcinoma available at our institution. Immunohistochemistry was performed for ST6GAL1 and percent positive cancer cell staining was assessed and correlation with pathological grade of response was measured. RESULTS: Organoid models were treated with chemoradiation and both ST6GAL1 mRNA and protein significantly increased after treatment. The organoid model targeted with ST6GAL1 knockdown was found to have increased CC3 after treatment. In the tissue microarray, 42 percent of patient samples had an increase in percent tumor cell staining for ST6GAL1 after treatment. Post-treatment percent staining was associated with a worse grade of treatment response (p = 0.01) and increased staining post-treatment compared to pre-treatment was also associated with a worse response (p = 0.01). CONCLUSION: ST6GAL1 is associated with resistance to treatment in human rectal cancer and knockdown in an organoid model abrogated resistance to apoptosis caused by chemoradiation.

Sialylation of EGFR by ST6GAL1 induces receptor activation and modulates trafficking dynamics.

Aberrant glycosylation is a hallmark of a cancer cell. One prevalent alteration is an enrichment in α2,6-linked sialylation of N-glycosylated proteins, a modification directed by the ST6GAL1 sialyltransferase. ST6GAL1 is upregulated in many malignancies including ovarian cancer. Prior studies have shown that the addition of α2,6 sialic acid to the epidermal growth factor receptor (EGFR) activates this receptor, although the mechanism was largely unknown. To investigate the role of ST6GAL1 in EGFR activation, ST6GAL1 was overexpressed in the OV4 ovarian cancer line, which lacks endogenous ST6GAL1, or knocked-down in the OVCAR-3 and OVCAR-5 ovarian cancer lines, which have robust ST6GAL1 expression. Cells with high expression of ST6GAL1 displayed increased activation of EGFR and its downstream signaling targets, AKT and NFκB. Using biochemical and microscopy approaches, including total internal reflection fluorescence microscopy, we determined that the α2,6 sialylation of EGFR promoted its dimerization and higher order oligomerization. Additionally, ST6GAL1 activity was found to modulate EGFR trafficking dynamics following EGF-induced receptor activation. Specifically, EGFR sialylation enhanced receptor recycling to the cell surface following activation while simultaneously inhibiting lysosomal degradation. 3D widefield deconvolution microscopy confirmed that in cells with high ST6GAL1 expression, EGFR exhibited greater colocalization with Rab11 recycling endosomes and reduced colocalization with LAMP1-positive lysosomes. Collectively, our findings highlight a novel mechanism by which α2,6 sialylation promotes EGFR signaling by facilitating receptor oligomerization and recycling.

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Sialylation of EGFR by ST6GAL1 induces receptor activation and modulates trafficking dynamics.

Aberrant glycosylation is a hallmark of a cancer cell. One prevalent alteration is an enrichment in α2,6-linked sialylation of N-glycosylated proteins, a modification directed by the ST6GAL1 sialyltransferase. ST6GAL1 is upregulated in many malignancies including ovarian cancer. Prior studies have shown that the addition of α2,6 sialic acid to the Epidermal Growth Factor Receptor (EGFR) activates this receptor, although the mechanism was largely unknown. To investigate the role of ST6GAL1 in EGFR activation, ST6GAL1 was overexpressed in the OV4 ovarian cancer line, which lacks endogenous ST6GAL1, or knocked down in the OVCAR-3 and OVCAR-5 ovarian cancer lines, which have robust ST6GAL1 expression. Cells with high expression of ST6GAL1 displayed increased activation of EGFR and its downstream signaling targets, AKT and NFκB. Using biochemical and microscopy approaches, including Total Internal Reflection Fluorescence (TIRF) microscopy, we determined that the α2,6 sialylation of EGFR promoted its dimerization and higher order oligomerization. Additionally, ST6GAL1 activity was found to modulate EGFR trafficking dynamics following EGF-induced receptor activation. Specifically, EGFR sialylation enhanced receptor recycling to the cell surface following activation while simultaneously inhibiting lysosomal degradation. 3D widefield deconvolution microscopy confirmed that in cells with high ST6GAL1 expression, EGFR exhibited greater co-localization with Rab11 recycling endosomes and reduced co-localization with LAMP1-positive lysosomes. Collectively, our findings highlight a novel mechanism by which α2,6 sialylation promotes EGFR signaling by facilitating receptor oligomerization and recycling.

Role of the ST6GAL1 sialyltransferase in regulating ovarian cancer cell metabolism.

The ST6GAL1 sialyltransferase, which adds α2-6-linked sialic acids to N-glycosylated proteins, is upregulated in many malignancies including ovarian cancer. Through its activity in sialylating select surface receptors, ST6GAL1 modulates intracellular signaling to regulate tumor cell phenotype. ST6GAL1 has previously been shown to act as a survival factor that protects cancer cells from cytotoxic stressors such as hypoxia. In the present study, we investigated a role for ST6GAL1 in tumor cell metabolism. ST6GAL1 was overexpressed (OE) in OV4 ovarian cancer cells, which have low endogenous ST6GAL1, or knocked-down (KD) in ID8 ovarian cancer cells, which have high endogenous ST6GAL1. OV4 and ID8 cells with modulated ST6GAL1 expression were grown under normoxic or hypoxic conditions, and metabolism was assessed using Seahorse technology. Results showed that cells with high ST6GAL1 expression maintained a higher rate of oxidative metabolism than control cells following treatment with the hypoxia mimetic, desferrioxamine (DFO). This enrichment was not due to an increase in mitochondrial number. Glycolytic metabolism was also increased in OV4 and ID8 cells with high ST6GAL1 expression, and these cells displayed greater activity of the glycolytic enzymes, hexokinase and phosphofructokinase. Metabolism maps were generated from the combined Seahorse data, which suggested that ST6GAL1 functions to enhance the overall metabolism of tumor cells. Finally, we determined that OV4 and ID8 cells with high ST6GAL1 expression were more invasive under conditions of hypoxia. Collectively, these results highlight the importance of sialylation in regulating the metabolic phenotype of ovarian cancer cells.

Role of tumor cell sialylation in pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies and is currently the third leading cause of cancer death. The aggressiveness of PDAC stems from late diagnosis, early metastasis, and poor efficacy of current chemotherapies. Thus, there is an urgent need for effective biomarkers for early detection of PDAC and development of new therapeutic strategies. It has long been known that cellular glycosylation is dysregulated in pancreatic cancer cells, however, tumor-associated glycans and their cognate glycosylating enzymes have received insufficient attention as potential clinical targets. Aberrant glycosylation affects a broad range of pathways that underpin tumor initiation, metastatic progression, and resistance to cancer treatment. One of the prevalent alterations in the cancer glycome is an enrichment in a select group of sialylated glycans including sialylated, branched N-glycans, sialyl Lewis antigens, and sialylated forms of truncated O-glycans such as the sialyl Tn antigen. These modifications affect the activity of numerous cell surface receptors, which collectively impart malignant characteristics typified by enhanced cell proliferation, migration, invasion and apoptosis-resistance. Additionally, sialic acids on tumor cells engage inhibitory Siglec receptors on immune cells to dampen anti-tumor immunity, further promoting cancer progression. The goal of this review is to summarize the predominant changes in sialylation occurring in pancreatic cancer, the biological functions of sialylated glycoproteins in cancer pathogenesis, and the emerging strategies for targeting sialoglycans and Siglec receptors in cancer therapeutics.

α2,6 Sialylation mediated by ST6GAL1 promotes glioblastoma growth.

One of the least-investigated areas of brain pathology research is glycosylation, which is a critical regulator of cell surface protein structure and function. ß-Galactoside α2,6-sialyltransferase (ST6GAL1) is the primary enzyme that α2,6 sialylates N-glycosylated proteins destined for the plasma membrane or secretion, thereby modulating cell signaling and behavior. We demonstrate a potentially novel, protumorigenic role for α2,6 sialylation and ST6GAL1 in the deadly brain tumor glioblastoma (GBM). GBM cells with high α2,6 sialylation exhibited increased in vitro growth and self-renewal capacity and decreased mouse survival when orthotopically injected. α2,6 Sialylation was regulated by ST6GAL1 in GBM, and ST6GAL1 was elevated in brain tumor-initiating cells (BTICs). Knockdown of ST6GAL1 in BTICs decreased in vitro growth, self-renewal capacity, and tumorigenic potential. ST6GAL1 regulates levels of the known BTIC regulators PDGF Receptor ß (PDGFRB), Activated Leukocyte Cell Adhesion Molecule, and Neuropilin, which were confirmed to bind to a lectin-recognizing α2,6 sialic acid. Loss of ST6GAL1 was confirmed to decrease PDGFRB α2,6 sialylation, total protein levels, and the induction of phosphorylation by PDGF-BB. Thus, ST6GAL1-mediated α2,6 sialylation of a select subset of cell surface receptors, including PDGFRB, increases GBM growth.

ST6Gal1: Oncogenic signaling pathways and targets.

The Golgi-sialyltransferase ST6Gal1 (ßgalactosidase α2,6 sialyltransferase 1), adds the negatively charged sugar, sialic acid, to the terminal galactose of N-glycosylated proteins. Upregulation of ST6Gal1 is observed in many malignancies, and a large body of research has determined that ST6Gal1-mediated α2,6 sialylation impacts cancer hallmarks. ST6Gal1 affects oncogenic behaviors including sustained proliferation, enhanced self-renewal, epithelial-to-mesenchymal transition, invasion, and chemoresistance. However, there are relatively few ST6GaL1 related signaling pathways that are well-established to mediate these biologies: greater delineation of specific targets and signaling mechanisms that are orchestrated by ST6Gal1 is needed. The aim of this review is to provide a summary of our current understanding of select oncogenic signaling pathways and targets affected by ST6Gal1.

The incorrect use of CD75 as a synonym for ST6GAL1 has fostered the expansion of commercial "ST6GAL1" antibodies that do not recognize ST6GAL1.

The ST6GAL1 Golgi sialyltransferase is upregulated in many human malignancies, however, detection of ST6GAL1 protein in cancer tissues has been hindered by the prior lack of antibodies. Recently, numerous commercial antibodies for ST6GAL1 have become available, however, many of these do not, in fact, recognize ST6GAL1. Decades ago, the CD75 cell-surface epitope was mistakenly suggested to be the same molecule as ST6GAL1. While this was rapidly disproven, the use of CD75 as a synonym for ST6GAL1 has persisted, particularly by companies selling "ST6GAL1" antibodies. CD75 is reportedly a sialylated epitope which appears to encompass a range of glycan structures and glycan carriers. In this study, we evaluated the LN1 and ZB55 monoclonal antibodies, which are advertised as ST6GAL1 antibodies but were initially developed as CD75-recognizing antibodies (neither was raised against ST6GAL1 as the immunogen). Importantly, the LN1 and ZB55 antibodies have been widely used by investigators, as well as the Human Protein Atlas database, to characterize ST6GAL1 expression. Herein, we used cell and mouse models with controlled expression of ST6GAL1 to compare LN1 and ZB55 with an extensively validated polyclonal antibody to ST6GAL1. We find that LN1 and ZB55 do not recognize ST6GAL1, and furthermore, these 2 antibodies recognize different targets. Additionally, we utilized the well-validated ST6GAL1 antibody to determine that ST6GAL1 is overexpressed in bladder cancer, a finding that contradicts prior studies which employed LN1 to suggest ST6GAL1 is downregulated in bladder cancer. Collectively, our studies underscore the need for careful validation of antibodies purported to recognize ST6GAL1.

ST6Gal-I-mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion.

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an 'allosteric mechano-organizer' of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against ß1, ß3, and ß5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.

Sialyltransferase ST6GAL-1 mediates resistance to chemoradiation in rectal cancer.

Locally advanced rectal cancer is typically treated with chemoradiotherapy followed by surgery. Most patients do not display a complete response to chemoradiotherapy, but resistance mechanisms are poorly understood. ST6GAL-1 is a sialyltransferase that adds the negatively charged sugar, sialic acid (Sia), to cell surface proteins in the Golgi, altering their function. We therefore hypothesized that ST6GAL-1 could mediate resistance to chemoradiation in rectal cancer by inhibiting apoptosis. Patient-derived xenograft and organoid models of rectal cancer and rectal cancer cell lines were assessed for ST6GAL-1 protein with and without chemoradiation treatment. ST6GAL-1 mRNA was assessed in untreated human rectal adenocarcinoma by PCR assays. Samples were further assessed by Western blotting, Caspase-Glo apoptosis assays, and colony formation assays. The presence of functional ST6GAL-1 was assessed via flow cytometry using the Sambucus nigra lectin, which specifically binds cell surface α2,6-linked Sia, and via lectin precipitation. In patient-derived xenograft models of rectal cancer, we found that ST6GAL-1 protein was increased after chemoradiation in a subset of samples. Rectal cancer cell lines demonstrated increased ST6GAL-1 protein and cell surface Sia after chemoradiation. ST6GAL-1 was also increased in rectal cancer organoids after treatment. ST6GAL-1 knockdown in rectal cancer cell lines resulted in increased apoptosis and decreased survival after treatment. We concluded that ST6GAL-1 promotes resistance to chemoradiotherapy by inhibiting apoptosis in rectal cancer cell lines. More research will be needed to further elucidate the importance and mechanism of ST6GAL-1-mediated resistance.

Regulation of ST6GAL1 sialyltransferase expression in cancer cells.

The ST6GAL1 sialyltransferase, which adds α2-6 linked sialic acids to N-glycosylated proteins, is overexpressed in a wide range of human malignancies. Recent studies have established the importance of ST6GAL1 in promoting tumor cell behaviors such as invasion, resistance to cell stress and chemoresistance. Furthermore, ST6GAL1 activity has been implicated in imparting cancer stem cell characteristics. However, despite the burgeoning interest in the role of ST6GAL1 in the phenotypic features of tumor cells, insufficient attention has been paid to the molecular mechanisms responsible for ST6GAL1 upregulation during neoplastic transformation. Evidence suggests that these mechanisms are multifactorial, encompassing genetic, epigenetic, transcriptional and posttranslational regulation. The purpose of this review is to summarize current knowledge regarding the molecular events that drive enriched ST6GAL1 expression in cancer cells.

Glycosyltransferase ST6Gal-I promotes the epithelial to mesenchymal transition in pancreatic cancer cells.

ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I's role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers (N-cadherin, slug, snail, fibronectin) and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers (E-cadherin, occludin) were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I's role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I's tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR.

Regulation of inflammatory signaling by the ST6Gal-I sialyltransferase.

The ST6Gal-I sialyltransferase, an enzyme that adds α2-6-linked sialic acids to N-glycosylated proteins, regulates multiple immunological processes. However, the contribution of receptor sialylation to inflammatory signaling has been under-investigated. In the current study, we uncovered a role for ST6Gal-I in promoting sustained signaling through two prominent inflammatory pathways, NFκB and JAK/STAT. Using the U937 monocytic cell model, we determined that knockdown (KD) of ST6Gal-I expression had no effect on the rapid activation of NFκB by TNF (≤ 30 min), whereas long-term TNF-induced NFκB activation (2-6 hr) was diminished in ST6Gal-I-KD cells. These data align with prior work in epithelial cells showing that α2-6 sialylation of TNFR1 prolongs TNF-dependent NFκB activation. Similar to TNF, long-term, but not short-term, LPS-induced activation of NFκB was suppressed by ST6Gal-I KD. ST6Gal-I KD cells also exhibited reduced long-term IRF3 and STAT3 activation by LPS. Given that ST6Gal-I activity modulated LPS-dependent signaling, we conducted pull-down assays using SNA (a lectin specific for α2-6 sialic acids) to show that the LPS receptor, TLR4, is a substrate for sialylation by ST6Gal-I. We next assessed signaling by IFNγ, IL-6 and GM-CSF, and found that ST6Gal-I-KD had a limited effect on STAT activation induced by these cytokines. To corroborate these findings, signaling was monitored in bone marrow derived macrophages (BMDMs) from mice with myeloid-specific deletion of ST6Gal-I (LysMCre/ST6Gal-Ifl/fl). In agreement with data from U937 cells, BMDMs with ST6Gal-I knockout displayed reduced long-term activation of NFκB by both TNF and LPS, and diminished long-term LPS-dependent STAT3 activation. However, STAT activation induced by IFNγ, IL-6 and GM-CSF was comparable in wild-type and ST6Gal-I knockout BMDMs. These results implicate ST6Gal-I-mediated receptor sialylation in prolonging the activity of select signaling cascades including TNF/NFκB, LPS/NFκB, and LPS/STAT3, providing new insights into ST6Gal-I's role in modulating the inflammatory phenotype of monocytic cells.

Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover.

Programmed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression levels of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis depended on ST6Gal-I overexpression, because forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of α2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate up-regulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.

3D printed mesh reinforcements enhance the mechanical properties of electrospun scaffolds.

BACKGROUND: There is substantial interest in electrospun scaffolds as substrates for tissue regeneration and repair due to their fibrous, extracellular matrix-like composition with interconnected porosity, cost-effective production, and scalability. However, a common limitation of these scaffolds is their inherently low mechanical strength and stiffness, restricting their use in some clinical applications. In this study we developed a novel technique for 3D printing a mesh reinforcement on electrospun scaffolds to improve their mechanical properties. METHODS: A poly (lactic acid) (PLA) mesh was 3D-printed directly onto electrospun scaffolds composed of a 40:60 ratio of poly(ε-caprolactone) (PCL) to gelatin, respectively. PLA grids were printed onto the electrospun scaffolds with either a 6 mm or 8 mm distance between the struts. Scanning electron microscopy was utilized to determine if the 3D printing process affected the archtitecture of the electrospun scaffold. Tensile testing was used to ascertain mechanical properties (strength, modulus, failure stress, ductility) of both unmodified and reinforced electrospun scaffolds. An in vivo bone graft model was used to assess biocompatibility. Specifically, reinforced scaffolds were used as a membrane cover for bone graft particles implanted into rat calvarial defects, and implant sites were examined histologically. RESULTS: We determined that the tensile strength and elastic modulus were markedly increased, and ductility reduced, by the addition of the PLA meshes to the electrospun scaffolds. Furthermore, the scaffolds maintained their matrix-like structure after being reinforced with the 3D printed PLA. There was no indication at the graft/tissue interface that the reinforced electrospun scaffolds elicited an immune or foreign body response upon implantation into rat cranial defects. CONCLUSION: 3D-printed mesh reinforcements offer a new tool for enhancing the mechanical strength of electrospun scaffolds while preserving the advantageous extracellular matrix-like architecture. The modification of electrospun scaffolds with 3D-printed reinforcements is expected to expand the range of clinical applications for which electrospun materials may be suitable.

Sox2 promotes expression of the ST6Gal-I glycosyltransferase in ovarian cancer cells.

BACKGROUND: The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to N-glycosylated proteins is upregulated in a wide range of malignancies including ovarian cancer. Prior studies have shown that ST6Gal-I-mediated sialylation of select surface receptors remodels intracellular signaling to impart cancer stem cell (CSC) characteristics. However, the mechanisms that contribute to ST6Gal-I expression in stem-like cancer cells are poorly understood. RESULTS: Herein, we identify the master stem cell transcription factor, Sox2, as a novel regulator of ST6Gal-I expression. Interestingly, SOX2 and ST6GAL1 are located within the same tumor-associated amplicon, 3q26, and these two genes exhibit coordinate gains in copy number across multiple cancers including ~ 25% of ovarian serious adenocarcinomas. In conjunction with genetic co-amplification, our studies suggest that Sox2 directly binds the ST6GAL1 promoter to drive transcription. ST6Gal-I expression is directed by at least four distinct promoters, and we identified the P3 promoter as the predominant promoter utilized by ovarian cancer cells. Chromatin Immunoprecipitation (ChIP) assays revealed that Sox2 binds regions proximal to the P3 promoter. To confirm that Sox2 regulates ST6Gal-I expression, Sox2 was either overexpressed or knocked-down in various ovarian cancer cell lines. Sox2 overexpression induced an increase in ST6Gal-I mRNA and protein, as well as surface α2-6 sialylation, whereas Sox2 knock-down suppressed levels of ST6Gal-I mRNA, protein and surface α2-6 sialylation. CONCLUSIONS: These data suggest a process whereby SOX2 and ST6GAL1 are coordinately amplified in cancer cells, with the Sox2 protein then binding the ST6GAL1 promoter to further augment ST6Gal-I expression. Our collective results provide new insight into mechanisms that upregulate ST6Gal-I expression in ovarian cancer cells, and also point to the possibility that some of the CSC characteristics commonly attributed to Sox2 may, in part, be mediated through the sialyltransferase activity of ST6Gal-I.

Sustained delivery of the angiogenic QK peptide through the use of polyglutamate domains to control peptide release from bone graft materials.

Angiogenesis plays a pivotal role in tissue regeneration following bone-grafting procedures; however, nonautogenous graft materials typically lack critical angiogenic growth factors. While much research has focused on modifying grafts with angiogenic factors, controlled delivery of these molecules remains a challenge. The current study describes a method for sustained delivery of an angiogenic peptide from hydroxyapatite (HA), a common alloplast material. Specifically, VEGF-derived "QK" peptides were synthesized with polyglutamate domains containing varying numbers of glutamates. The rate of peptide release from HA inversely correlated with glutamate number, with diglutamate-QK (E2-QK) released first, followed by tetraglutamate-QK (E4-QK), and finally, heptaglutamate-QK (E7-QK). By coating HA with a mixture of these peptides, termed, PGM-QK (polyglutamate-modified mixture), sequential peptide release was achieved, enabling gradient QK delivery. To evaluate bioactivity, HA disks were coated with PGM-QK and then placed in fresh media for 6 days. Media containing the released peptides was collected at varying time intervals and placed on human umbilical vein endothelial cells (HUVECs). Cells were evaluated for activation of angiogenic signaling pathways (ERK and Akt) and cell migration. Results showed that QK peptides were continuously released over the 6-day interval, and maintained their capacity to activate HUVECs. These findings point to a new approach for gradient delivery of an angiogenic stimulus.