Globo-series Glycosphingolipids Research Articles

Regulation of Glycosylation in Bone Metabolism.

Glycosylation plays a crucial role in the maintenance of homeostasis in the body and at the onset of diseases such as inflammation, neurodegeneration, infection, diabetes, and cancer. It is also involved in bone metabolism. N- and O-glycans have been shown to regulate osteoblast and osteoclast differentiation. We recently demonstrated that ganglio-series and globo-series glycosphingolipids were essential for regulating the proliferation and differentiation of osteoblasts and osteoclasts in glycosyltransferase-knockout mice. Herein, we reviewed the importance of the regulation of bone metabolism by glycoconjugates, such as glycolipids and glycoproteins, including our recent results.

Increased expression of SSEA-4 on TKI-resistant non–small cell lung cancer with EGFR-T790M mutation

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme β3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Priming of macrophage by glycosphingolipids from extracellular vesicles facilitates immune tolerance for embryo-maternal crosstalk

Glycosphingolipids (GSLs) display diverse functions during embryonic development. Here, we examined the GSL profiles of extracellular vesicles (EVs) secreted from human embryonic stem cells (hESCs) and investigated their functions in priming macrophages to enhance immune tolerance of embryo implantation. When peripheral blood mononuclear cells were incubated with ESC-secreted EVs, globo-series GSLs (GHCer, SSEA3Cer, and SSEA4Cer) were transferred via EVs into monocytes/macrophages. Incubation of monocytes during their differentiation into macrophages with either EVs or synthetic globo-series GSLs induced macrophages to exhibit phenotypic features that imitate immune receptivity, i.e., macrophage polarization, augmented phagocytic activity, suppression of Tcell proliferation, and the increased trophoblast invasion. It was also demonstrated that decidual macrophages in first-trimester tissues expressed globo-series GSLs. These findings highlight the role of globo-series GSLs via transfer from EVs in priming macrophages to display decidual macrophage phenotypes, which may facilitate healthy pregnancy.

A concise chemoenzymatic total synthesis of neutral Globo-series glycosphingolipids Globo A and Globo B, and Forssman and para-Forssman antigens.

Globo A is a neutral Globo-series glycosphingolipid (GSL) that shows natural properties of a cytotoxicity receptor NKp44 binding ligand. The highly complex heptasaccharide glycan structure of GloboA combined with its biological profile provides a unique target for the development of a synthetic method to facilitate its bioactivity studies. Here, a concise chemoenzymatic route to the synthesis of Globo A and its α1,3-galactose-linked congener Globo B is reported. The key to success was the use of a synthetic azido β-Globo H sphingosine (Globo H-βSph) as an acceptor substrate and two glycosyl transferases, an α1,3-N-acetylgalactosaminyltransferase from Helicobactermustelae (BgtA) and a human blood group B α1,3-galactosyltransferase (h1,3GTB), for stereoselective construction of the terminal α1,3-GalNAc and α1,3-Gal linkages, respectively. The azido-Sph lipid sidechain is further elaborated by reduction and a chemoselective N-acylation to complete the total synthesis of the neutral Globo-series GSLs. In addition, the synthesis of Forssman and para-Forssman antigens were prepared. The strategy may be suitable for accessing other complex GSLs and related lipid-modified GSL derivatives.

Recently developed glycosphingolipid probes and their dynamic behavior in cell plasma membranes as revealed by single-molecule imaging.

Glycosphingolipids, including gangliosides, are representative lipid raft markers that perform a variety of physiological roles in cell membranes. However, studies aimed at revealing their dynamic behavior in living cells are rare, mostly due to a lack of suitable fluorescent probes. Recently, the ganglio-series, lacto-series, and globo-series glycosphingolipid probes, which mimic the behavior of the parental molecules in terms of partitioning to the raft fraction, were developed by conjugating hydrophilic dyes to the terminal glycans of glycosphingolipids using state-of-art entirely chemical-based synthetic techniques. High-speed, single-molecule observation of these fluorescent probes revealed that gangliosides were scarcely trapped in small domains (100nm in diameter) for more than 5ms in steady-state cells, suggesting that rafts including gangliosides were always moving and very small. Furthermore, dual-color, single-molecule observations clearly showed that homodimers and clusters of GPI-anchored proteins were stabilized by transiently recruiting sphingolipids, including gangliosides, to form homodimer rafts and the cluster rafts, respectively. In this review, we briefly summarize recent studies, the development of a variety of glycosphingolipid probes as well as the identification of the raft structures including gangliosides in living cells by single-molecule imaging.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Abstract 4084: Globo H-targeted CAR T cell cancer immunotherapy

Abstract Background: Globo H (GH), a Globo-series glycosphingolipid (GSL), is highly expressed in epithelial tumors, such as colon, endometrial, gastric, pancreatic, lung, prostate, and breast cancers. Aberrant expression of Globo H has been reported to be associated with the metastatic potential and poor prognosis of these cancers. However, in normal tissues, GH expression is limited to the secretory borders of apical epithelial cells, making it difficult to access by the immune system. GH is therefore a promising target for anticancer therapeutics. Clinical studies with the Globo H vaccines (OBI-822 and OBI-833) and the humanized anti-Globo H antibody (OBI-888) demonstrating an excellent safety profile. In recent years, cell therapy using chimeric antigen receptor T (CAR T) cells have shown impressive clinical outcomes. As a result, development of CAR T targeting GH may offer a novel anticancer therapeutic agent. Aim: The aim of this study was to develop a CAR T cell therapy targeted against Globo H (obi-R007) using lentivirus-mediated genetic engineering with a proprietary Globo H-specific antibody. Methods: We conducted in vitro and in vivo studies to determine the characteristics of GH CAR T obi-R007. Results: Ex vivo expansion of obi-R007 from healthy donors ranged from 40- to 200-fold post-activation for 10 days. Previous studies have suggested that less differentiation and fewer exhaustion markers of CAR T cells are beneficial for therapeutic persistence. In our study, there was less differentiation in the CD62L+ population (TSCM and TCM) in greater than 80% of obi-R007 cells, and no significant exhaustion markers (PD-1, LAG-3, etc.) were detected. obi-R007 showed specific cytotoxicity against Globo H-positive tumor cells with an E/T ratio of 0.5:1~2:1 and exhibited Globo H-specific activation through the expression of CD69, CD25, and Granzyme B as well as the release of Interferon γ and IL-2. Moreover, the activation and proliferation of obi-R007 were dependent on the presence of target cells. All the in vitro results indicate the specific targeting of obi-R007 to Globo H resulting in cytotoxic T cell responses. In the in vivo study, the efficacy of obi-R007 was demonstrated by adoptive cell transfer in several xenograft models and obi-R007 was shown to be persistent under multiple tumor challenges in the NCI-N87 gastric cancer model. Luminance labelling of CAR T cells showed that the distribution of CAR T cells in mice was specific to the tumor site and lymphoid organs suggesting a homing activity of the CAR T cells. Furthermore, no obviously physiological toxicity was observed in vivo. Conclusion: In conclusion, our in vitro and in vivo pharmacology and preliminary toxicology studies support clinical development of Globo H CAR T immunotherapy for patients with cancer. Citation Format: Jiann-Shiun Lai, Shiou-Ling Jian, Woan-Eng Chan, Ming-Tain Lai. Globo H-targeted CAR T cell cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4084.

Abstract 6138: High Globo-H expression associated with poor survival of gastric cancer patients and enriched PD-L1 expression

Abstract Background: Globo series antigens have been implicated in playing important roles in enhancing tumor growth through protecting tumor cells from apoptosis, suppressing T cell activity in the tumor microenvironment and promoting endothelial cell angiogenesis. Globo-H (GH), a globo-series glycosphingolipid (GSL) antigen that is synthesized by key enzymes β1,3-galactosyltransferase V (β3GalT5), fucosyltransferase (FUT) 1 and 2, is highly expressed on a variety of epithelial cancers rendering it a promising target for cancer immunotherapy. GH is reported to associate the EGFR mutant and PD-L1 expression in non-small cell lung cancer (NSCLC) patients. In addition, GH-targeting antibody-drug conjugate (ADC) OBI-999 has been demonstrated an excellent tumor growth inhibition potency in animal models across multiple cancer types including gastric cancer (GC). This study aims to further investigate the GH roles in GC. Methods: Prognostic roles of β3GalT5, FUT1 and FUT2 in GC patients were accessed through the “Kaplan-Meier plotter” database. The level of GH expression was evaluated in clinical adenocarcinoma samples from 105 patients with GC by immunohistochemistry (IHC) using H-score. Microwestern protein array analysis was performed in sorted GC NCI-N87 cells. FACS methods were also employed to measure the levels of PD-L1 and phospho-EGFRs in multiple cancer cell lines. Results: Significant correlations were observed between high mRNA expression of synthesized key enzymes and worse overall survival (OS)/post-progression survival for all 498 GC patients. Positive GH expression (H score≥20; 33.3%) was significantly associated with a poor disease specific survival in all samples (at 15 years; p=0.029), a poor OS in poorly differentiated tumors (p=0.033), and invasiveness (p=0.013), indicating GH expression was a potential prognostic factor in GC. Sorted NCI-N87 cells with high level of endogenous GH expression showed a relatively greater proliferative activity compared with cells expressing low level of GH. Microwestern array analysis on sorted cells indicated upregulations of phospho-AKT/P38/JNK, Cyclin D1 and Cyclin E1 protein in high GH expression NCI-N87 cells, which implicated a mechanistic link between high GH and tumor promoting signals in GC. In addition, GH level was shown to be correlated with cell surface expression level of PD-L1 in NCI-N87, SNU-16, HCC-1428, ACHN, and TKI-resistant H1975 NSCLC cancer cells. Higher phospho-EGFRs (Tyr1068 and Tyr1173) were detected in high GH level of H1975 cells, which is consistent with the results from previous studies showing association between GH level and EGFR mutant/PD-L1 expression in NSCLC. Conclusions: GH level was associated with the survival of GC patients and positively correlated with cell surface PD-L1 expression in vitro. Therefore, GH-targeting therapy has a potential application for the treatment of GC patients. Citation Format: Tzer-Min Kuo, Hui-Ting Hsu, Cheng-Yen Wei, Ming-Tain Lai, Chun-Te Chen. High Globo-H expression associated with poor survival of gastric cancer patients and enriched PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6138.

Abstract P5-08-01: Globo H: A Globo series glycosphingolipid target antigen in breast cancer

Abstract Cancer cells may be differentiated from normal cells by the surface expression of unusual glycosylation patterns: tumor-associated carbohydrate antigens (TACAs) may be expressed as membrane-bound glycoproteins or glycolipids. Differential expression of TACAs in multiple cancer types represents a potential therapeutic target for anticancer therapies. At least 16 different types of epithelial cancers have been found to be Globo H–positive, with 60% to 65% of patients with breast cancer having Globo H–positive tumors. It has been shown that tumor-infiltrating lymphocytes in breast cancer tissues could take up Globo H ceramide (GHCer) released by tumor cells masking cancer cells from immune surveillance through the suppression of Notch 1 signaling. Globo series antigens, including Globo H and stage-specific embryonic antigen 4 could associate with the FAK/CAV1/AKT/RIP complex to trigger the downstream β-catenin signaling pathway to promote tumor cell survival. Moreover, exogenous addition of GHCer promoted tumor cell migration. Thus, treatment-mediated depletion of GHCer may overcome the immunosuppressive mechanism of cancer cells, with resulting decreases in tumor cell survival and migration. OBI Pharma, Inc., in collaboration with NeoGenomics, has developed a validated immunohistochemistry assay to measure the expression of Globo H in clinical specimens. The intensity and extent of Globo H expression are determined microscopically using the histochemical scoring system (H-score), defined as the sum of the products of the staining intensity (score of 0-3) multiplied by the percentage of cells (0-100) stained at a given intensity. To date, 420 patients with triple-negative breast cancer (TNBC) have been screened across 4 separate clinical trials. Of those patients, 173 (41%) had an H-score of ≥15, with a median H-score of 70. Of the 173 patients with an H-score of ≥15, 69 (40%) had an H-score of ≥100. Adagloxad simolenin (AS/OBI-822) is a Globo H conjugate vaccine that when administered in combination with the adjuvant OBI-821 results in anti–Globo H IgM and IgG humoral immune responses. These responses have been assessed in all clinical trials of AS/OBI-821 to date. In the early trials that administered up to 9 doses, early and robust IgM responses were noted but with delayed and lower-titer IgG responses. When the number of doses was increased to 9-12 doses over an extended time (up to either 37 or 68 weeks), it became apparent that the peak IgG titers occurred at around the time of the last AS/OBI-821 dose. The time course for the development of anti–Globo H IgM and IgG humoral responses showed that following injection, anti–Globo H IgM and IgG antibody titers demonstrated a geometric mean ratio (GMR) increase of 10.26 and 12.47, respectively, from almost nondetectable levels at baseline. The GMR in the placebo group remained low in comparison. Mean time to peak titer was approximately 4 weeks for IgM and 40 weeks for IgG. These data were employed in the design of the phase 3, randomized, open-label study of the anti–Globo H vaccine AS/OBI-821 in the adjuvant treatment of patients with high-risk, early-stage Globo H–positive TNBC (The GLORIA Study). To enter the GLORIA study, patients must have a centrally confirmed Globo H IHC H-score of ≥15, and it is anticipated that approximately 40% of patients screened will have this level of expression. The GLORIA study is presently enrolling patients in 7 countries, with another 9 countries anticipated to begin recruiting in 2021. Citation Format: Darren S. Sigal, Apostolia Maria Tsimberidou, I-Ju Chen, Pei Hsu, Tillman Pearce. Globo H: A Globo series glycosphingolipid target antigen in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-01.

Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx.

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.

Combined Effect of Anti-SSEA4 and Anti-Globo H Antibodies on Breast Cancer Cells.

The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an ∼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.

Abstract 3176: Inhibitory activity of Globo-H and SSEA-4 on activated T-cells

Abstract Background/Aim: Globo-H (GH) and stage-specific embryonic antigen-4 (SSEA-4) are globo-series glycosphingolipid (GSL) antigens highly expressed on a variety of epithelial cancers. GH and SSEA-4 are reported to play an important role in cancer cell development and survival and thus are promising targets for developing immunotherapeutic agents for cancer treatment. Shedding of GSLs by tumor cells and uptake by neighboring cells may generate a preferable environment for tumor progression and survival within the tumor microenvironment. GH shed from tumor cells was shown to promote angiogenesis and suppress immune responses in vitro. In addition, exogenous Globo-H ceramide (GHCer) inhibits proliferation of human PBMCs and murine CD4+ T-cells. Given the important role of sialic acid in regulating the immune system, SSEA-4, a polysaccharide with sialic acid at the terminal, may play an important role in the regulation of T-cell responses. This study aimed to investigate the GHCer and SSEA-4 ceramide (SSEA-4Cer) effects on T-cell activation. Methods: C57BL/6 mice bearing murine B16F10 melanoma were treated with GHCer by peritumoral injection and monitored for 26 days. T-cell tumor subpopulations were evaluated by FACS and immunohistochemical analysis. Presence of GHCer and SSEA-4Cer expression on human T cells after the treatment with GHCer and SSEA-4Cer was monitored by FACS analysis. Cell proliferation and apoptosis of T cells after treatment with GHCer, SSEA-4Cer, and glucoceramide (GlcCer) were assessed using ATP assay and annexin V staining, respectively. After treatment with GHCer/SSEA-4Cer/GlcCer, expression levels of CD25, cytokine IFN-γ, and Ki-67 in human PBMCs stimulated with anti-CD3/CD28 were measured by flow cytometry. Results: B16F10 tumor growth rate increased 2.54-fold (P<0.05, n=6) in GHCer-treated mice with a lower number of tumor-infiltrating CD3+ cells (0.45-fold; P<0.01, n=6) compared with control mice. Not surprisingly, addition of GHCer and SSEA-4Cer to human T cells was shown to increase GH and SSEA-4 levels on the surface of T cells. Furthermore, addition of GHCer and SSEA-4Cer to the activated T cells showed a 26.4% and 36.9% decrease in cell proliferation, respectively, while no induction of apoptosis was observed. In contrast, GHCer had no impact on cell viability and proliferation of resting T cells. Exogenous GHCer and SSEA-4Cer significantly reduced CD3+CD25+ and CD3+IFNγ+ T cells in anti-CD3/CD28-stimulated PBMCs. SSEA-4Cer exhibited higher activity in the suppression of CD8+CD25+ T cells in PBMCs than GHCer did. In addition, suppression of the proliferation of activated T-cells by SSEA-4Cer was abolished in the presence of NF-κB inhibitor. Conclusion: GHCer inhibits T-cell infiltration into the tumor in vivo and SSEA-4 exhibits immunosuppressive activity on activated T cells, suggesting that both GHCer and SSEA-4Cer may play a role in regulating immunologic responses in the tumor microenvironment. Citation Format: Tzer-Min Kuo, Chin-Chan Lee, Jiann-Shiun Lai, Chung-Chen Su, Ming-Tain Lai. Inhibitory activity of Globo-H and SSEA-4 on activated T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3176.

Abstract 2934: The role of Globo H in cancer cell survival

Abstract BACKGROUND: Globo H (GH), a glycosphingolipid (GSL) carbohydrate antigen, is found on a variety of epithelial tumors and is a promising target for cancer immunotherapy given the marked difference in expression of GH between cancer cells and normal tissues. Globo series antigens (Globo H, SSEA-4) have been implicated in playing important roles in protecting tumor cells from apoptosis, suppression of immune cell activity in the tumor microenvironment, and promoting endothelial cell angiogenesis through shedding the glycosphingolipids to immune cells and endothelial cells, respectively. Recently, apoptosis regulation by protein interaction with Globo-series GSLs in breast cancer cells was reported. This study aims to investigate the GH and anti-GH antibody effects on cancer cell survival. METHODS: In vivo: After allografting murine B16F10 melanoma subcutaneously, C57BL/6 mice, n=5, were treated with Globo-H ceramide (GHCer) by peritumoral injection and monitored for 26 days. In vitro: GH expression on the surface of tumor cells and colony formative activity in GHCer-treated human cells were measured. Cytokine antibody array and Microwestern protein array analyses were performed in the GHCer-treated A549 cells. Cell viability in high GH–expressing lung cancer cells treated with erlotinib was assessed. Erlotinib resistance and cytokine levels in high GH–expressing tumor cells treated with anti-GH antibody were measured. RESULTS: In vivo: GHCer-treated mice showed accelerated B16F10-tumor growth rates (3.36-fold; P<0.01, n=5) with less tumor necrosis and higher proliferating cell nuclear antigen expression in the internal tumor tissues. In vitro: In GHCer-treated human lung cancer A549 and breast cancer MDA-MB-231 cells, higher colony formative activity and increased exogenous GH expression on the surface of tumor cells compared to the control group were observed, while cell proliferation was not enhanced. Up-regulated erlotinib-resistant cytokine IL-8 and FGF2 and K-RAS-related signaling were observed in A549 cells with exogenous high GH expression. Exogenously GHCer-added lung cancer cells displayed higher cell viability (37.8% increase) during erlotinib challenge, and an anti-GH antibody treatment decreased the GH effects of erlotinib resistance. CONCLUSION: GH promotes cancer cell viability in vitro and in vivo, indicating an important role in cancer cell survival during tumor progression. Anti-GH antibody treatment was shown to be able to abolish the GH effects on erlotinib resistance of lung cancer cells in vitro. Citation Format: Tzer-Min Kuo, Yi-Chien Tsai, Chin-Chan Lee, Jiann-Shiun Lai. The role of Globo H in cancer cell survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2934.

Globo-series glycosphingolipids deficiency in mice resulted in the attenuation of bone formation through decrease of osteoblasts

Globo-series glycosphingolipids deficiency in mice resulted in the attenuation of bone formation through decrease of osteoblasts

Deletion of Gb3 Synthase in Mice Resulted in the Attenuation of Bone Formation via Decrease in Osteoblasts

Glycosphingolipids are known to play a role in developing and maintaining the integrity of various organs and tissues. Among glycosphingolipids, there are several reports on the involvement of gangliosides in bone metabolism. However, there have been no reports on the presence or absence of expression of globo-series glycosphingolipids in osteoblasts and osteoclasts, and the involvement of their glycosphingolipids in bone metabolism. In the present study, we investigated the presence or absence of globo-series glycosphingolipids such as Gb3 (globotriaosylceramide), Gb4 (globoside), and Gb5 (galactosyl globoside) in osteoblasts and osteoclasts, and the effects of genetic deletion of Gb3 synthase, which initiates the synthesis of globo-series glycosphingolipids on bone metabolism. Among Gb3, Gb4, and Gb5, only Gb4 was expressed in osteoblasts. However, these glycosphingolipids were not expressed in pre-osteoclasts and osteoclasts. Three-dimensional micro-computed tomography (3D-μCT) analysis revealed that femoral cancellous bone mass in Gb3 synthase-knockout (Gb3S KO) mice was lower than that in wild type (WT) mice. Calcein double labeling also revealed that bone formation in Gb3S KO mice was significantly lower than that in WT mice. Consistent with these results, the deficiency of Gb3 synthase in mice decreased the number of osteoblasts on the bone surface, and suppressed mRNA levels of osteogenic differentiation markers. On the other hand, osteoclast numbers on the bone surface and mRNA levels of osteoclast differentiation markers in Gb3S KO mice did not differ from WT mice. This study demonstrated that deletion of Gb3 synthase in mice decreases bone mass via attenuation of bone formation.

PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1-4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer), globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) and Forssman GSL (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) revealed balanced adhesion toward the three GSLs for PapG I-mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III-harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface-exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

Signaling pathway of globo-series glycosphingolipids and β1,3-galactosyltransferase V (β3GalT5) in breast cancer

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme β1,3-galactosyltransferase V (β3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of β3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of β3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

Globo-series glycosphingolipids enhance Toll-like receptor 4-mediated inflammation and play a pathophysiological role in diabetic nephropathy.

Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inflammatory processes are involved in development and progression of diabetic nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles in inflammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role of these GSLs in this disease process. We studied proinflammatory activity of GSLs in diabetic nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD) (60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer), upregulation of TNF-α, and induction of renal inflammation. Gb3Cer/Gb4Cer treatment enhanced inflammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T, mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our findings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inflammatory response, and that such GSLs play an important pathophysiological role in diabetic nephropathy.

α-galactosidase A deficiency promotes von Willebrand factor secretion in models of Fabry disease

Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in invivo and invitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both invitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease.

Glycosphingolipid metabolic reprogramming drives neural differentiation.

Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of thefirst and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo-AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.