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.

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.

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.

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.

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.

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.

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.

The ST6Gal-I sialyltransferase protects tumor cells against hypoxia by enhancing HIF-1α signaling.

Aberrant cell surface glycosylation is prevalent in tumor cells, and there is ample evidence that glycans have functional roles in carcinogenesis. Nonetheless, many molecular details remain unclear. Tumor cells frequently exhibit increased α2-6 sialylation on N-glycans, a modification that is added by the ST6Gal-I sialyltransferase, and emerging evidence suggests that ST6Gal-I-mediated sialylation promotes the survival of tumor cells exposed to various cell stressors. Here we report that ST6Gal-I protects cancer cells from hypoxic stress. It is well known that hypoxia-inducible factor 1α (HIF-1α) is stabilized in hypoxic cells, and, in turn, HIF-1α directs the transcription of genes important for cell survival. To investigate a putative role for ST6Gal-I in the hypoxic response, we examined HIF-1α accumulation in ovarian and pancreatic cancer cells in ST6Gal-I overexpression or knockdown experiments. We found that ST6Gal-I activity augmented HIF-1α accumulation in cells grown in a hypoxic environment or treated with two chemical hypoxia mimetics, deferoxamine and dimethyloxalylglycine. Correspondingly, hypoxic cells with high ST6Gal-I expression had increased mRNA levels of HIF-1α transcriptional targets, including the glucose transporter genes GLUT1 and GLUT3 and the glycolytic enzyme gene PDHK1 Interestingly, high ST6Gal-I-expressing cells also had an increased pool of HIF-1α mRNA, suggesting that ST6Gal-I may influence HIF-1α expression. Finally, cells grown in hypoxia for several weeks displayed enriched ST6Gal-I expression, consistent with a pro-survival function. Taken together, these findings unravel a glycosylation-dependent mechanism that facilitates tumor cell adaptation to a hypoxic milieu.

ST6Gal-I sialyltransferase promotes tumor necrosis factor (TNF)-mediated cancer cell survival via sialylation of the TNF receptor 1 (TNFR1) death receptor.

Activation of the tumor necrosis factor receptor 1 (TNFR1) death receptor by TNF induces either cell survival or cell death. However, the mechanisms mediating these distinct outcomes remain poorly understood. In this study, we report that the ST6Gal-I sialyltransferase, an enzyme up-regulated in numerous cancers, sialylates TNFR1 and thereby protects tumor cells from TNF-induced apoptosis. Using pancreatic and ovarian cancer cells with ST6Gal-I knockdown or overexpression, we determined that α2-6 sialylation of TNFR1 had no effect on early TNF-induced signaling events, including the rapid activation of NF-κB, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt (occurring within 15 min). However, upon extended TNF treatment (6-24 h), cells with high ST6Gal-I levels exhibited resistance to TNF-induced apoptosis, as indicated by morphological evidence of cell death and decreased activation of caspases 8 and 3. Correspondingly, at these later time points, high ST6Gal-I expressers displayed sustained activation of the survival molecules Akt and NF-κB. Additionally, extended TNF treatment resulted in the selective enrichment of clonal variants with high ST6Gal-I expression, further substantiating a role for ST6Gal-I in cell survival. Given that TNFR1 internalization is known to be essential for apoptosis induction, whereas survival signaling is initiated by TNFR1 at the plasma membrane, we examined TNFR1 localization. The α2-6 sialylation of TNFR1 was found to inhibit TNF-induced TNFR1 internalization. Thus, by restraining TNFR1 at the cell surface via sialylation, ST6Gal-I acts as a functional switch to divert signaling toward survival. These collective findings point to a novel glycosylation-dependent mechanism that regulates the cellular response to TNF and may promote cancer cell survival within TNF-rich tumor microenvironments.

ST6Gal-I sialyltransferase promotes chemoresistance in pancreatic ductal adenocarcinoma by abrogating gemcitabine-mediated DNA damage.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis. Gemcitabine, as a single agent or in combination therapy, remains the frontline chemotherapy despite its limited efficacy due to de novo or acquired chemoresistance. There is an acute need to decipher mechanisms underlying chemoresistance and identify new targets to improve patient outcomes. Here, we report a novel role for the ST6Gal-I sialyltransferase in gemcitabine resistance. Utilizing MiaPaCa-2 and BxPC-3 PDAC cells, we found that knockdown (KD) of ST6Gal-I expression, as well as removal of surface α2-6 sialic acids by neuraminidase, enhances gemcitabine-mediated cell death assessed via clonogenic assays and cleaved caspase 3 expression. Additionally, KD of ST6Gal-I potentiates gemcitabine-induced DNA damage as measured by comet assays and quantification of γH2AX foci. ST6Gal-I KD also alters mRNA expression of key gemcitabine metabolic genes, RRM1, RRM2, hENT1, and DCK, leading to an increased gemcitabine sensitivity ratio, an indicator of gemcitabine toxicity. Gemcitabine-resistant MiaPaCa-2 cells display higher ST6Gal-I levels than treatment-naïve cells along with a reduced gemcitabine sensitivity ratio, suggesting that chronic chemotherapy selects for clonal variants with more abundant ST6Gal-I. Finally, we examined Suit2 PDAC cells and Suit2 derivatives with enhanced metastatic potential. Intriguingly, three metastatic and chemoresistant subclones, S2-CP9, S2-LM7AA, and S2-013, exhibit up-regulated ST6Gal-I relative to parental Suit2 cells. ST6Gal-I KD in S2-013 cells increases gemcitabine-mediated DNA damage, indicating that suppressing ST6Gal-I activity sensitizes inherently resistant cells to gemcitabine. Together, these findings place ST6Gal-I as a critical player in imparting gemcitabine resistance and as a potential target to restore PDAC chemoresponse.

The Glycosyltransferase ST6Gal-I Protects Tumor Cells against Serum Growth Factor Withdrawal by Enhancing Survival Signaling and Proliferative Potential.

A hallmark of cancer cells is the ability to survive and proliferate when challenged with stressors such as growth factor insufficiency. In this study, we report a novel glycosylation-dependent mechanism that protects tumor cells from serum growth factor withdrawal. Our results suggest that the ß-galactoside α-2,6-sialyltransferase 1 (ST6Gal-I) sialyltransferase, which is up-regulated in numerous cancers, promotes the survival of serum-starved cells. Using ovarian and pancreatic cancer cell models with ST6Gal-I overexpression or knockdown, we find that serum-starved cells with high ST6Gal-I levels exhibit increased activation of prosurvival signaling molecules, including pAkt, p-p70S6K, and pNFκB. Correspondingly, ST6Gal-I activity augments the expression of tumor-promoting pNFκB transcriptional targets such as IL-6, IL-8, and the apoptosis inhibitor cIAP2. ST6Gal-I also potentiates expression of the cell cycle regulator cyclin D2, leading to increased phosphorylation and inactivation of the cell cycle inhibitor pRb. Consistent with these results, serum-starved cells with high ST6Gal-I expression maintain a greater number of S phase cells compared with low ST6Gal-I expressors, reflecting enhanced proliferation. Finally, selective enrichment in clonal variants with high ST6Gal-I expression is observed upon prolonged serum deprivation, supporting the concept that ST6Gal-I confers a survival advantage. Collectively, these results implicate a functional role for ST6Gal-I in fostering tumor cell survival within the serum-depleted tumor microenvironment.

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.

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.

Regulation of the metastatic cell phenotype by sialylated glycans.

Tumor cells exhibit striking changes in cell surface glycosylation as a consequence of dysregulated glycosyltransferases and glycosidases. In particular, an increase in the expression of certain sialylated glycans is a prominent feature of many transformed cells. Altered sialylation has long been associated with metastatic cell behaviors including invasion and enhanced cell survival; however, there is limited information regarding the molecular details of how distinct sialylated structures or sialylated carrier proteins regulate cell signaling to control responses such as adhesion/migration or resistance to specific apoptotic pathways. The goal of this review is to highlight selected examples of sialylated glycans for which there is some knowledge of molecular mechanisms linking aberrant sialylation to critical processes involved in metastasis.

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.

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.

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.

Emerging role of alpha2,6-sialic acid as a negative regulator of galectin binding and function.

Galectins are ß-galactoside-binding lectins that regulate diverse cell behaviors, including adhesion, migration, proliferation, and apoptosis. Galectins can be expressed both intracellularly and extracellularly, and extracellular galectins mediate their effects by associating with cell-surface oligosaccharides. Despite intensive current interest in galectins, strikingly few studies have focused on a key enzyme that acts to inhibit galectin signaling, namely ß-galactoside α2,6-sialyltransferase (ST6Gal-I). ST6Gal-I adds an α2,6-linked sialic acid to the terminal galactose of N-linked glycans, and this modification blocks galectin binding to ß-galactosides. This minireview summarizes the evidence suggesting that ST6Gal-I activity serves as an "off switch" for galectin function.

Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells.

The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.