Lewis Y Research Articles

Vaccination with DC-SIGN-Targeting αGC Liposomes Leads to Tumor Control, Irrespective of Suboptimally Activated T-Cells.

Cancer vaccines have emerged as a potent strategy to improve cancer immunity, with or without the combination of checkpoint blockade. In our investigation, liposomal formulations containing synthetic long peptides and α-Galactosylceramide, along with a DC-SIGN-targeting ligand, Lewis Y (LeY), were studied for their anti-tumor potential. The formulated liposomes boosted with anti-CD40 adjuvant demonstrated robust invariant natural killer (iNKT), CD4+, and CD8+ T-cell activation in vivo. The incorporation of LeY facilitated the targeting of antigen-presenting cells expressing DC-SIGN in vitro and in vivo. Surprisingly, mice vaccinated with LeY-modified liposomes exhibited comparable tumor reduction and survival rates to those treated with untargeted counterparts despite a decrease in antigen-specific CD8+ T-cell responses. These results suggest that impaired induction of antigen-specific CD8+ T-cells via DC-SIGN targeting does not compromise anti-tumor potential, hinting at alternative immune activation routes beyond CD8+ T-cell activation.

α1,3-fucosylation of MEST promotes invasion potential of cytotrophoblast cells by activating translation initiation

Embryo implantation into the uterus is the gateway for successful pregnancy. Proper migration and invasion of embryonic trophoblast cells are the key for embryo implantation, and dysfunction causes pregnancy failure. Protein glycosylation plays crucial roles in reproduction. However, it remains unclear whether the glycosylation of trophoblasts is involved in trophoblast migration and invasion processes during embryo implantation failure. By Lectin array, we discovered the decreased α1,3-fucosylation, especially difucosylated Lewis Y (LeY) glycan, in the villus tissues of miscarriage patients when compared with normal pregnancy women. Downregulating LeY biosynthesis by silencing the key enzyme fucosyltransferase IV (FUT4) inhibited migration and invasion ability of trophoblast cells. Using proteomics and translatomics, the specific LeY scaffolding glycoprotein of mesoderm-specific transcript (MEST) with glycosylation site at Asn163 was identified, and its expression enhanced migration and invasion ability of trophoblast cells. The results also provided novel evidence showing that decreased LeY modification on MEST hampered the binding of MEST with translation factor eIF4E2, and inhibited implantation-related gene translation initiation, which caused pregnancy failure. The α1,3-fucosylation of MEST by FUT4 may serve as a new biomarker for evaluating the functional state of pregnancy, and a target for infertility treatment.

Sialyl Lewis X mediates interleukin-1 beta-induced trophoblast adhesion to endometrial cells during human embryo implantation.

Cell surface carbohydrate antigens sialyl Lewis X (sLeX) and Lewis Y (LeY) are paramount glycoconjugates and are abundantly expressed in the receptive endometrium. Furthermore, among the important biological functions of both antigens is their role in leukocytes adhesion and extravasation. Interleukin-1 beta (IL-1β) is involved in the process of human embryo implantation and placenta development. Here, we used an in vitro model to investigate whether sLeX and LeY are playing a role in the embryo implantation process mediated by IL-1β. Our results are showing that the expression of cell surface sLeX was enhanced in endometrial RL95-2 cells after exposure to IL-1β. RT-qPCR detection indicated that the transcript level of glycosyltransferase gene fucosyltransferase 3 (FUT3) was significantly elevated and that of FUT4/7 and ST3 beta-galactoside alpha-2,3-sialyltransferase 3/4 (ST3GAL3/4) were decreased by treatment with IL-1β. Modulatory role of glycosyltransferase FUT3 on sLeX biosynthesis was determined by FUT3 siRNA transfection in RL95-2 cells. Results showed that the expression level of sLeX was suppressed, but no change was observed in regard to LeY. Moreover, IL-1β promoted the HTR-8/SVneo trophoblast spheroids attachment to the RL95-2 endometrial monolayer, which was partially blocked by anti-sLeX antibody and FUT3 knockdown. Gene expression analysis of the RNA-seq transcriptome data from human secretory endometrium demonstrated a significantly higher level of FUT3 in the mid-secretory phase compared to the early secretory phase, which was correlated with the expression of IL1B. In summary, the inflammatory microenvironment at the fetomaternal interface can regulate the glycosylation pattern of endometrial cells at the time of implantation. SLeX can be significantly induced by IL-1β via increasing FUT3 expression, which facilitates the trophoblast adhesion during embryo implantation.

Harnessing Antibody Polyspecificity for Cancer Immunotherapy.

Targeting the diverse glycan repertoire expressed on tumor cells is considered a viable therapeutic strategy to deal with tumor cell heterogeneity. Inherently polyspecific, natural, glycan-reactive antibodies are purported to be protective in thwarting infections and in cancer immunotherapy. Tumor-associated carbohydrate antigens (TACAs) are related to pathogen glycans, to which nascent or natural antibodies exist and IgM responses are elicited. To capture the polyspecific nature of anticarbohydrate responses, we have focused on the rational design of carbohydrate mimetic peptides (CMPs) cross-reactive with TACA reactive antibodies. In particular, we have focused on the development of CMPs that display reactivity to GD2 and Lewis Y (LeY) reactive monoclonal antibodies. They would serve as templates for pan-immunogens inducing biosimilar polyreactive antibodies. In the design, we relied on structural analyses of CMP's enhanced binding to the templates using molecular modeling. Glycan reactivity patterns of affinity CMP-purified human antibodies further refined specificity profiles in comparison with the immune response to the CMP in clinical trials. In this study, we further define the molecular characteristics for this mimicry by considering the polyspecificity of LeY and GD2 reactive antibodies binding to the lacto-ceramide core Galβ(1,4)Glcβ(1-1')Cer. Binding to this minimum building block can be capitalized on for cancer therapy and diagnostics and illustrates a new approach in designing cancer vaccines taking advantage of the latent polyspecificity of antibodies and the relevance of natural antibodies in antigen discovery and design.

Targeting Aberrantly Elevated Sialyl Lewis A as a Potential Therapy for Impaired Endometrial Selection Ability in Unexplained Recurrent Miscarriage.

BackgroundCarbohydrate Lewis antigens including sialyl Lewis A (sLeA), sialyl Lewis X (sLeX), Lewis X (LeX), and Lewis Y (LeY) are the commonest cell surface glycoconjugates that play pivotal roles in multiple biological processes, including cell adhesion and cell communication events during embryogenesis. SLeX, LeY, and associated glycosyltransferases ST3GAL3 and FUT4 have been reported to be involved in human embryo implantation. While the expression pattern of Lewis antigens in the decidua of unexplained recurrent miscarriage (uRM) patients remains unclear.MethodsParaffin-embedded placental tissue slides collected from patients experiencing early miscarriages (6–12 weeks) were analyzed using immunohistochemical (IHC) and immunofluorescent (IF) staining. An in vitro assay was developed using endometrial cell line RL95-2 and trophoblast cell line HTR-8/SVneo. Modulatory effect of potential glycosyltransferase on Lewis antigens expression was investigated by target-specific small interfering RNA (siRNA) knockdown in RL95-2 cells. HTR-8/SVneo cells spheroids adhesion assay was applied to investigate the intrinsic role of Lewis antigens in the abnormal implantation process of uRM. The expression of Lewis antigens in RL95-2 cells in response to the treatment with pro-implantation cytokine IL-1β was further measured by flow cytometry and immunocytochemical (ICC) staining.ResultsIHC staining revealed that Lewis antigens are mainly expressed in the luminal and glandular epithelium, IF staining further indicated the cellular localization at the apical membrane of the epithelial cells. FUTs, ST3GALs, and NEU1 located in both stromal and epithelial cells. We have found that the expression of sLeA, LeX, FUT3/4, and ST3GAL3/4 are significantly upregulated in the RM group, while FUT1 is downregulated. SLeX, LeY, ST3GAL6, and NEU1 showed no significant differences between groups. FUT3 knockdown in RL95-2 cells significantly decreased the expression of sLeA and the spheroids adhesion to endometrial monolayer. Anti-sLeA antibody can remarkably suppress both the basal and IL-1β induced adhesion of HTR-8/SVneo spheroids to RL95-2 cells monolayer. While further flow cytometry and ICC detection indicated that the treatment of RL95-2 cells with IL-1β significantly increases the surface expression of LeX, but not sLeA.ConclusionsSLeA, LeX, and pertinent glycosyltransferase genes FUT1/3/4 and ST3GAL3/4 are notably dysregulated in the decidua of uRM patients. FUT3 accounts for the synthesis of sLeA in RL95-2 cells and affects the endometrial receptivity. Targeting aberrantly elevated sLeA may be a potential therapy for the inappropriate implantation in uRM.

Blood group antigens SLeX, SLeA, and LeY as prognostic markers in endometrial cancer

PurposeEndometrial cancer (EC) is the most common gynecological cancer worldwide. Treatment has been improved in recent years, but, in advanced stages, therapeutical options are still limited. It has been reported that the expression of the blood group antigens Sialyl Lewis X (SLeX), Sialyl Lewis A (SLeA) and Lewis Y (LeY) is associated with prognosis in several tumors. Large studies on endometrial and cervical cancer are still pending.MethodsSpecimens of 234 patients with EC were immunohistochemically stained with antibodies for SLeX, SLeA and LeY. Expression was correlated to histopathological variables.ResultsHigh expression of SLeX was correlated to low pT-stage (p = 0.013), low grade (p < 0.001), low FIGO-stage (p = 0.006) and better overall survival rates (OS; p = 0.023). High expression of SLeA was associated with low pT-stage (p = 0.013), low grade (p = 0.001) and better progression-free survival (PFS; p = 0.043). LeY staining was correlated to pN + (p = 0.038), low grade (p = 0.005) and poorer PFS (p = 0.022).ConclusionThis is the first study examining the expression of SLeX, SLeA and LeY in EC, which can serve as additional future prognostic markers. Further studies are necessary to understand the underlying mechanisms. The study was approved by the local ethics committee of the Ludwig-Maximilians University Munich (reference number 19-249).

Abstract 2400: Fucosyltransferase expression is associated with head and neck cancer survival

Abstract Introduction: Head and neck (H&amp;N) cancers account for 5-7% of new cancer cases in the United States, and most manifest as head and neck squamous cell carcinomas (HNSCC). Fucosyltransferases (FUTs) contribute to the formation of sialofucosylated epitopes, which alter cellular signaling and mediate early steps in metastasis. However, few studies have examined the effect of FUT expression on H&amp;N cancer survival. Here, we sought to examine the association between fucosyltransferase expression and H&amp;N survival and investigate the mechanistic role of fucosylation in H&amp;N cancer. Methods: FUT1-11 expression (mRNA) data was extracted from the Cancer Genome Atlas (TCGA) HNSCC dataset (n=499). Kaplan-Meier, logrank, and Cox proportional hazards tests were conducted. Levels of α1,2-fucosylated epitopes H-antigen, Lewis Y (LeY), and Lewis B (LeB) were measured in normal, dysplastic, and H&amp;N tumor cell lines via flow cytometry, and RT-qPCR was performed to assess FUT expression. To identify fucosylated signaling and adhesion molecules, fucosylated glycopeptides were enriched from CAL27 and HSC-3 tongue squamous cell carcinoma (SCC) lysates via AAL lectin, followed by nLC-MS/MS. EGFR signaling was assessed via western blot, and parallel plate flow assays were performed to assess in vitro metastatic potential, in tumor cell lines with differential FUT expression. Results: In TCGA analyses, the median survival time (MST) of the FUT2 high expression group was 4.7 years, while low expression group MST was 2.7 years, with an adjusted hazard ratio (aHR) of 0.72 (CI = 0.54-0.95). The FUT6-high group MST was 4.7 years versus 2.9 years for low expression (aHR = 0.62, CI = 0.47-0.83). The FUT7-high group MST was 4.7 years versus 3.5 years for low expression (aHR = 0.72, CI = 0.54-0.95). Expression of the LeY epitope, with both α1,2- and α1,3-linked fucose, varied widely in tongue SCC lines, while pharyngeal SCC lines FaDu and Det-562 displayed intermediate LeY levels, via flow cytometry. EGFR, CD44, and integrins, were among the fucosylated glycoproteins identified via mass spectrometry. HSC-3 cells, sorted into low- and high-LeY populations, demonstrated differential EGFR Tyr-1086 phosphorylation, and differences in adhesion to endothelial cells under flow conditions (in vitro), with implications for tumor metastasis. Conclusion: Overexpression of FUT2, FUT6, and FUT7 were associated with statistically significant increases in survivorship. FUT6 and FUT7 are prominently expressed in immune cells, while FUT2 is expressed in endoderm-derived epithelium. Therefore, FUT6 and FUT7 expression may indicate immune cell infiltration into tumor tissue. Future studies will seek to sort tumor tissue based on cell type, to gain an understanding of the cell-type specific glycosyltransferase expression in HNSCC tumors. Our current results will serve as the foundation to interrogate the role of fucosylated glycoproteins in HNSCC metastasis. Citation Format: Kevin Brown Chandler, Brittany Montesino, Nan Hu, Juan M. Lozano, Robert Sackstein. Fucosyltransferase expression is associated with head and neck cancer survival [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 2400.

Expression of the Carbohydrate Lewis Antigen, Sialyl Lewis A, Sialyl Lewis X, Lewis X, and Lewis Y in the Placental Villi of Patients With Unexplained Miscarriages.

BackgroundLewis antigens such as Sialyl Lewis A (sLeA), Sialyl Lewis X (sLeX), Lewis X (LeX), and Lewis Y (LeY) are a class of carbohydrate molecules that are known to mediate adhesion between tumor cells and endothelium by interacting with its selectin ligands. However, their potential role in miscarriage remains enigmatic. This study aims to analyze the expression pattern of sLeA, sLeX, LeX, and LeY in the placental villi tissue of patients with a medical history of unexplained miscarriages.MethodsParaffin-embedded slides originating from placental tissue were collected from patients experiencing a miscarriage early in their pregnancy (6–13 weeks). Tissues collected from spontaneous (n = 20) and recurrent (n = 15) miscarriages were analyzed using immunohistochemical and immunofluorescent staining. Specimens obtained from legally terminated normal pregnancies were considered as control group (n = 18). Assessment of villous vessel density was performed in another cohort (n = 10 each group) of gestation ages-paired placenta tissue. Protein expression was evaluated with Immunoreactive Score (IRS). Statistical analysis was performed by using Graphpad Prism 8.ResultsExpression of sLeA, sLeX, LeX, and LeY in the syncytiotrophoblast was significantly upregulated in the control group compared with spontaneous and recurrent miscarriage groups. However, no prominent differences between spontaneous and recurrent miscarriage groups were identified. Potential key modulators ST3GAL6 and NEU1 were found to be significantly downregulated in the recurrent miscarriage group and upregulated in the spontaneous group, respectively. Interestingly, LeX and LeY expression was also detected in the endothelial cells of villous vessels in the control group but no significant expression in miscarriage groups. Furthermore, assessment of villous vessel density using CD31 found significantly diminished vessels in all size groups of villi (small villi <200 µm, P = 0.0371; middle villi between 200 and 400 µm, P = 0.0010 and large villi >400 µm, P = 0.0003). Immunofluorescent double staining also indicated the co-localization of LeX/Y and CD31.ConclusionsThe expression of four mentioned carbohydrate Lewis antigens and their potential modulators, ST3GAL6 and NEU1, in the placenta of patients with miscarriages was significantly different from the normal pregnancy. For the first time, their expression pattern in the placenta was illustrated, which might shed light on a novel understanding of Lewis antigens’ role in the pathogenesis of miscarriages.

Investigating Conformational Dynamics of Lewis Y Oligosaccharides and Elucidating Blood Group Dependency of Cholera Using Molecular Dynamics.

Cholera is caused by Vibrio cholerae and is an example of a blood-group-dependent disease. Recent studies suggest that the receptor-binding B subunit of the cholera toxin (CT) binds histo–blood group antigens at a secondary binding site. Herein, we studied the conformational dynamics of Lewis Y (LeY) oligosaccharides, H-tetrasaccharides and A-pentasaccharides, in aqueous solution by conducting accelerated molecular dynamics (aMD) simulations. The flexible nature of both oligosaccharides was displayed in aMD simulations. Furthermore, aMD simulations revealed that for both oligosaccharides in the free form, 4C1 and 1C4 puckers were sampled for all but GalNAc monosaccharides, while either the 4C1 (GlcNAc, Gal, GalNAc) or 1C4 (Fuc2, Fuc3) pucker was sampled in the CT-bound forms. In aMD, the complete transition from the 4C1 to 1C4 pucker was sampled for GlcNAc and Gal in both oligosaccharides. Further, we have observed a transition from the open to closed conformer in the case of A-pentasaccharide, while H-tetrasaccharide remains in the open conformation throughout the simulation. Both oligosaccharides adopted an open conformation in the CT binding site. Moreover, we have investigated the molecular basis of recognition of LeY oligosaccharides by the B subunit of the cholera toxin of classical and El Tor biotypes using the molecular mechanics generalized Born surface area (MM/GBSA) scheme. The O blood group determinant, H-tetrasaccharide, exhibits a stronger affinity to both biotypes compared to the A blood group determinant, A-pentasaccharide, which agrees with the experimental data. The difference in binding free energy between O and A blood group determinants mainly arises due to the increased entropic cost and desolvation energy in the case of A-pentasaccharide compared to that of H-tetrasaccharide. Our study also reveals that the terminal Fuc3 contributes most to the binding free energy compared to other carbohydrate residues as it forms multiple hydrogen bonds with CT. Overall, our study might help in designing glycomimetic drugs targeting the cholera toxin.

Expression of fucosylated glycans in endothelial glycocalyces of placental villi at early and late fetal growth restriction

The aim of the study was to investigate the content and distribution of fucosylated sugar residues and Lewis Y (LeY) in the endothelial glycocalyx (eGC) in placental tissue at early and late onset fetal growth restriction (FGR). Our findings demonstrated that the changes of the fucosylated glycans of type 2 (H2)/LeY in the vascular endothelium of the villi may reflect alteration of villi maturation, or adaptation to hypoxia through the change of cell proliferation potential and induction angiogenesis. Early onset FGR differs from late onset FGR by a markedly increased LeY expression, being associated with more severe pathological state.

Revisit ROS in Early Pregnancy: Essential for Placentation but Quantity Matters

The aim of the study was to investigate the content and distribution of fucosylated sugar residues and Lewis Y (LeY) in the endothelial glycocalyx (eGC) in placental tissue at early and late onset fetal growth restriction (FGR). Our findings demonstrated that the changes of the fucosylated glycans of type 2 (H2)/LeY in the vascular endothelium of the villi may reflect alteration of villi maturation, or adaptation to hypoxia through the change of cell proliferation potential and induction angiogenesis. Early onset FGR differs from late onset FGR by a markedly increased LeY expression, being associated with more severe pathological state.

Development of next generation car's targeting the lewis y antigen for the treatment of cancer

Background & Aim Refractory solid tumors remain an ongoing clinical problem with significant morbidity and mortality. CAR T cell therapy has the potential to address this issue. However, in contrast to their success in B cell malignancies, CAR T cell therapy targeting solid cancer antigens has had limited success so far due to several reasons including tumor antigen heterogeneity. To address this issue, we have previously generated a second-generation CAR recognizing the carbohydrate antigen Lewis Y (LeY). Given that LeY is highly expressed in various epithelial cancers but appears to have only limited expression on the surface of normal tissues making this antigen an ideal target for cancer immunotherapy. In a Phase I clinical trial involving transfer of anti-LeY CAR T cells in AML patients the detection and monitoring of CAR T cells in peripheral blood was challenging. To overcome this problem we have generated a retroviral vector containing the anti-LeY CAR, and a truncated CD34 gene. Methods, Results & Conclusion We successfully generated a stably expressing packaging cell line for this anti-LeY-CAR-CD34 containing retroviral vector. Retroviral supernatant derived from these packaging cells was then used to successfully transduce primary human T cells with the anti-LeY CAR and positive cells were sorted by flow cytometry using the CD34 marker. After sorting by flow cytometry, the majority of the T cells (>90%) were double positive for the anti-LeY CAR and CD34 marker. Antigen-specific function of the gene-modified T cells was then examined in cytokine and cytotoxicity assays to examine whether the presence of the CD34 marker may have compromised CAR function. The anti-LeY-CD34 CAR-T cells were found to secrete high levels of interferon γ, TNFα and IL-2 following direct stimulation through the CAR using immobilized plate bound anti-idiotype antibody or LeY+ tumor targets. The CAR T cells also specifically killed LeY+ tumor cells but not LeY− targets cells indicating that functional responses were not affected by the presence of the CD34 marker. Our results suggest that these new hu-CD34 CARs against LeY tumor antigen could offer new-targeted therapy with acceptable toxicity for patients with solid cancers given the low expression of this antigen on normal tissues. Furthermore this innovative tracking system with the CD34 marker will allow better characterisation and monitoring of the infused CAR-T cells in vivo in preclinical mouse models and in patients in future trials.

The content of immunomodulatory glycoepitopes in seminal plasma glycoproteins of fertile and infertile men.

According to a concept of fetoembryonic defence, protein-carbohydrate interaction may be involved in the regulation of maternal immunity that prevents rejection of allograft spermatozoa, embryo and fetus. In the present study we focussed on the evaluation of the expression of glycoepitopes that may be of crucial importance in this process: LewisY (LeY) and LewisX (LeX) as well as terminal sialylation. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate was used to separate seminal plasma samples of fertile (n=10) and infertile (n=103) men; these were then probed with lectins specific to fucose (Lotus tetragonolobus agglutinin and Ulex europaeus agglutinin) and sialic acid (Sambucus nigra agglutinin and Maackia amurensis agglutinin). Differential expression of α2,3-bound sialic acid was found in six out of seven analysed bands, whereas differences in the other analysed glycoepitopes were found in fewer numbers of bands. Mass spectrometry analysis focussed on the identification of proteins carrying glycans with immunomodulatory epitopes, including fibronectin, lactoferrin, clusterin, zinc-α2-glycoprotein, prostate acid phosphatase and prostate-specific antigen; these should be submitted to further detailed analysis.

Immune responses against Lewis Y tumor-associated carbohydrate antigen displayed densely on self-assembling nanocarriers.

Immune responses against Lewis y (LY) displayed on nanocarriers at different surface densities were studied. The high surface density of LY was obtained by the A2B-type amphiphilic polypeptides having LY at the two terminals [LY-poly(sarcosine)2-b-(l- or d-Leu-Aib)6]. The equimolar mixture of these two amphiphilic polypeptides formed interdigitated planar sheet-like molecular assemblies densely displaying LY (G4). G4 seemed to induce the anti-LY IgM upon immunization to BALB/c mice by only a single administration. However, the amount of anti-LY IgM produced was moderate and significantly less than that induced by two administrations of the other molecular assembly (G1) with the average surface density of LY at a 1/4 of that of G4. Further, the anti-LY IgM produced after two administrations of G4 lowered the avidity more than after one administration.

Targeting tumor-associated carbohydrate antigens: a phase I study of a carbohydrate mimetic-peptide vaccine in stage IV breast cancer subjects.

Tumor-associated carbohydrate antigens (TACAs) support cell survival that could be interrupted by anti-TACA antibodies. Among TACAs that mediate cell survival signals are the neolactoseries antigen Lewis Y (LeY) and the ganglioside GD2. To induce sustained immunity against both LeY and GD2, we developed a carbohydrate mimicking peptide (CMP) as a surrogate pan-immunogen that mimics both. This CMP, referred to as P10s, is the N-terminal half of a peptide vaccine named P10s-PADRE, the C-terminal half of which (PADRE) is a Pan-T-cell epitope. A Phase I dose-escalation trial of P10s-PADRE plus adjuvant MONTANIDE™ ISA 51 VG was conducted in subjects with metastatic breast cancer to test 300 and 500 μg/injection in two cohorts of 3 subjects each. Doses of the P10s-PADRE vaccine were administered to research participants subcutaneously on weeks 1, 2, 3, 7 and 19. Antibody responses to P10s, GD2, and LeY were measured by ELISA. The P10s-PADRE vaccine induced antibodies specifically reactive with P10s, LeY and GD2 in all 6 subjects. Serum antibodies displayed Caspase-3-dependent apoptotic functionality against LeY or GD2 expressing breast cancer cell lines. Immunization with the P10s-PADRE vaccine was well-tolerated and induced functional antibodies, and the data suggest potential clinical benefit.

MiR-200b inhibits proliferation and metastasis of breast cancer by targeting fucosyltransferase IV and \u03b11,3-fucosylated glycans

Aberrant protein fucosylation is associated with cancer malignancy. Fucosyltransferase IV (FUT4) is the key enzyme catalyzing the biosynthesis of α1,3-linkage fucosylated glycans carried by glycoproteins on the cell surface, such as the tumor-associated sugar antigen Lewis Y (LeY). An abnormal increase in the levels of FUT4 and LeY is observed in many cancers and correlated with cell proliferation and metastasis. Some microRNAs (miRNAs) are known to negatively regulate gene expression. FUT4 is an oncogenic glycogene, and thus it is important to identify the specific miRNA targeting FUT4. In current study, we first identified miR-200b as a specific miRNA that inhibited FUT4 expression. We found that miR-200b level was decreased, whereas that of FUT4 was increased in tissues and serum of breast cancer compared with that in the control by real-time PCR, western blotting and enzyme-linked immunosorbent assay. The alterations of miR-200b and FUT4 level were recovered after chemotherapy. The results also showed that miR-200b suppressed FUT4 expression and inhibited tumor growth and metastasis in MCF-7 and MDA-MB-231 breast cancer cells, as well as in the xenografted tumor tissues and metastatic lung tissues. miR-200b decreased the α1,3-fucosylation and LeY biosynthesis on epidermal growth factor receptor (EGFR), as well as inactivation of EGFR and downstream phosphoinositide-3 kinase/Akt signaling pathway. In conclusion, the study highlights that FUT4 could apply as a novel target for miR-200b that suppress the proliferation and metastasis of breast cancer cells by reducing α1,3-fucosylation and LeY biosynthesis of glycoproteins. miR-200b and FUT4 are potential diagnostic and therapeutic targets for breast cancer.

Anti-LeY monoclonal antibody (mAb) hu3S193 as consolidation therapy for patients (pts) with relapsing platinum sensitive ovarian cancer (OC) after achieving a second complete response (2CR).

e17039 Background: Lewis-Y (LeY) antigen is a blood group related antigen expressed in 75% of OC. hu3S193 is a humanized anti-LeY IgG1 mAb with strong complement and antibody dependent cytotoxicity with clinical benefit shown in a phase II study in pts with platinum-resistant OC and small tumor burden. Improving progression free survival (PFS) in pts who achieve a 2CR is an unmet need. Methods: This phase II study accrued pts with relapsed OC, LeY expression by IHC, and a KPS ≥ 70% who had achieved a 2CR after 5-8 cycles of platinum doublet chemotherapy (chemoRx). Patients received intravenous infusions of hu3S193, 30mg/m2every 2 weeks starting ≤ 8 weeks after the last dose of chemoRx and continuing for 12 doses (24 weeks) or until disease progression or unacceptable toxicity. Primary endpoint was PFS with a sample size calculated to detect a 50% increase in PFS, compared to a historical value of 10.8 months. Secondary objectives were safety and pharmacokinetics (PK). Results: 29 pts were enrolled. Median age: 55 yrs (range 29-67); Median KPS: 90% (range 80-100); LeY expression by IHC (N): strong (20), weak (9); stage at initial diagnosis: I/II (2), III/IV (27); median CA-125 prior to 2CR chemoRx: 104 (range 9-514); chemoRx agents to achieve 2CR: paclitaxel/platin (21), liposomal doxo/carboplatin (8); median cycles of prior chemoRx to achieve a 2CR:6; median doses of hu3S193 delivered: 10 (range 10-12). Evaluable pts: 28 (1 pt did not have 2CR); median PFS: 11.8 months (95% CI: 10.6-13.9) while 3 pts achieved PFS of 25+ months. Grade 4 toxicities observed: none. Most frequent toxicities (any grade/grade 3): Nausea (16/2), Vomiting (15/3), Hypersensitivity (9/0). PK data will be presented. Conclusions: Despite the good tolerance and the longer PFS compared to historical control, this trial did not show a significant improvement with hu3S193 as a consolidative strategy in patients with 2CR in platinum-sensitive OC. Tumor molecular analysis of patients with long PFS may provide insight for future studies. Clinical trial information: NCT01137071.

Tumor-associated macrophages promote Ezrin phosphorylation-mediated epithelial-mesenchymal transition in lung adenocarcinoma through FUT4/LeY up-regulation.

Tumor-associated macrophages (TAMs) are key components of tumor microenvironment (TME) during tumorigenesis and progression. However, the role of TAMs in lung adenocarcinoma is still unclear. In this study, we aimed to clarify the mechanism underlying the crosstalk between TAMs and epithelial-mesenchymal transition (EMT) of lung adenocarcinoma. Fucosyltransferase IV (FUT4) and its synthetic cancer sugar antigen Lewis Y (LeY) was aberrantly elevated in various solid tumors, it plays critical role in the invasion and metastasis. Here, we found that in lung adenocarcinoma samples, the density of TAMs correlates with E-cadherin level and LeY level. In vitro assays, M2 macrophages promoted FUT4/LeY expression through the transforming growth factor-β1(TGF-β1)/Smad2/3 signaling pathway. FUT4/LeY was indispensable in M2 macrophages-mediated cytoskeletal remodeling and EMT. Furthermore, fucosylation of Ezrin mediated by FUT4/LeY can promote the phosphorylation of Ezrin, which was the critical mechanism of M2 macrophages-induced EMT. In vivo assays confirmed that M2 macrophages promoted EMT through the up-regulation of LeY and phosphorylated Ezrin. Together, our results revealed that TAMs promote Ezrin phosphorylation-mediated EMT in lung adenocarcinoma through FUT4/LeY- mediated fucosylation. Targeting this newly identified signaling may offer new possibilities for immunotherapy in lung adenocarcinoma.

Sandwich ELISA Using a Mouse/Human Chimeric CSLEX-1 Antibody.

An assay using a mouse antisialyl Lewis X (sLeX) antibody (CSLEX-1) is used clinically for screening and monitoring patients with breast cancer in Japan. However, the IgM isoform of CSLEX-1 is not preferred for the assay because the bulkiness of IgM generally causes poor accessibility to the antigen. To solve this problem, we developed an antisLeX mouse/human chimeric IgG antibody, CH-CSLEX-1, using transgenic silkworms. The performance of a homologous sandwich ELISA of CH-CSLEX1 was then evaluated. To generate CH-CSLEX-1, we used a GAL4/UAS binary gene expression system in transgenic silkworms. The reactivities of CSLEX-1 and CH-CSLEX-1 were determined in a Biacore analysis. To confirm antigen specificity, 3 antigens [sLeX, sLeA, and Lewis Y (LeY)] were used. CH-CSLEX-1 formed correctly as an IgG class of immunoglobulin molecule with an isoelectric point close to the predicted value. The best combination for capturing and probing in a sandwich ELISA was determined as a homologous combination of CH-CSLEX-1. The CH-CSLEX-1 assay specifically detected sLeX, but not sLeA and LeY. A correlation analysis with 107 human samples showed good concordance between the conventional CSLEX-1 assay (homologous sandwich ELISA using CSLEX-1) and the CH-CSLEX-1 assay (r = 0.98). Moreover, the CH-CSLEX-1 assay was not affected by either human antimouse IgG antibodies (HAMA IgG) or HAMA IgM. The mouse/human chimeric antibody CH-CSLEX-1 allowed the establishment of a highly specific sandwich ELISA for sLeX that was not affected by HAMA.

Fucosyltransferase-4 and Oligosaccharide Lewis Y Antigen as potentially Correlative Biomarkers of Helicobacter pylori CagA Associated Gastric Cancer.

H. pylori cytotoxin associated antigen A (CagA) plays a significant role in the progression of gastric cancer but their effect on fucosylation to develop gastric cancer is unknown. Fucosyltransferase IV (FUT4) is the key enzyme for synthesis of LewisY (LeY) carried by glycoproteins and glycolipids on the cell membrane. Herein, we compare the expression of CagA, p-EGFR, FUT4 and LeY in gastritis (n=128, 176), gastric ulcer (n=174, 213), and gastric cancer (n=323, 261) tissue and serum samples, respectively by IHC and ELISA. Moreover, we investigated the potential correlation of CagA with FUT4 and LeY overexpression through EGFR activation. IHC and ELISA results showed higher positive cases of H. pylori CagA (83, 86 %), p-EGFR (81, 72 %), FUT4 (91, 97%) and LeY (93, 92 %) in gastric cancer, compared to gastritis and gastric ulcer, H. pylori CagA (58, 67 & 59, 73 %), p-EGFR (52, 63 & 35, 47 %), FUT4 (68, 78 & 67, 82 %) and LeY (62,76 & 65, 85 %), respectively. We found a significant high expression (H-Value) of CagA (1.79, 1.66), p-EGFR (1.53, 1.58), FUT4 (2.14, 1.66) and LeY (1.69, 1.61) in gastric cancer tissues and serum, respectively as compared to chronic gastritis and gastric ulcers, CagA (0.64,1.14), p-EGFR (0.856, 0.678), FUT4 (0.949,1.197) and LeY (0.68,1.008) (P<0.0001), respectively. Furthermore, H. pylori CagA showed significant correlation with p-EGFR (R-0.62, -0.74), FUT4 (R-0.81, -0.76) and LeY (R-0.82, -0.70) in gastric tissues and serum (P<0.0001). H. pylori CagA plays key role in the development of gastric cancer with overexpression of FUT4/LeY, serve as potentially correlative biomarkers of H. pylori CagA associated gastric cancer.