Fucosylation of LAMP-1 and LAMP-2 by FUT1 correlates with lysosomal positioning and autophagic flux of breast cancer cells.

Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. Lysosome-associated membrane protein 1, LAMP-1, has been reported to carry alpha1,2-fucosylated Lewis Y (LeY) antigens in breast cancer cells, however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member, LAMP-2, displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and 2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS(3) and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER- breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple-negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mammalian target of rapamycin complex 1 (mTORC1) and enhanced autophagosome-lysosome fusion. This may be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased rate of autophagic flux by decreasing mTOR signaling and increasing autolysosome formation

Smaller size packs a stronger punch - Recent advances in small antibody fragments targeting tumour-associated carbohydrate antigens

Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human cells. Unique glycan structures are monitored by the immune system and differentiate self from non-self and healthy from malignant cells. Aberrant glycosylations, termed tumour-associated carbohydrate antigens (TACAs), are a hallmark of cancer and are correlated with all aspects of cancer biology. Therefore, TACAs represent attractive targets for monoclonal antibodies for cancer diagnosis and therapy. However, due to the thick and dense glycocalyx as well as the tumour micro-environment, conventional antibodies often suffer from restricted access and limited effectiveness in vivo. To overcome this issue, many small antibody fragments have come forth, showing similar affinity with better efficiency than their full-length counterparts. Here we review small antibody fragments against specific glycans on tumour cells and highlight their advantages over conventional antibodies

Targeting aberrantly elevated Sialyl Lewis A as a potential therapy for impaired endometrial selection ability in unexplained recurrent miscarriage

BACKGROUND: Carbohydrate 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. METHODS: Paraffin-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. RESULTS: IHC 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. CONCLUSIONS: SLeA, 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

Monoclonal Antibodies Against Tumour-Associated Carbohydrate Antigens

Glycomic profiling of tumour tissues consistently shows alterations in N- and O-glycosylation profiles of glycoproteins and glycolipids compared to healthy tissues, with important functional implications for cancer cell biology. The overexpression of tumour-associated carbohydrate antigens (TACAs), as a result of aberrant glycosylation in tumours, is usually correlated with poor prognosis and survival of cancer patients. In tumours, TACAs are associated with worse tumour progression than the deletion and inactivation of tumour suppressor genes. The findings of TACAs acting are not merely tumour markers but also constitute part of the machinery in inducing cancer metastasis and invasiveness further strengthen the scientific rationales for immunotherapy targeting TACAs. Despite the attractiveness of the TACAs, there are very few anti-glycan monoclonal antibodies (mAbs), as glycans usually induce low-affinity IgM responses. This chapter provides an overview of TACAs, direct killing anti-glycan mAbs, and introduces two murine mAbs (FG88 mAbs) that recognise Lewis carbohydrate antigens overexpressed on tumour glycoconjugates with high functional affinity. Although the production of anti-glycan mAbs against cancers is not new, the production of high-affinity IgG anti-glycan mAbs is novel. FG88 mAbs definitely have great potential in cancer therapy and serve as valuable tools in glycobiology research

Fucosyltransferase 1 and 2 play pivotal roles in breast cancer cells.

FUT1 and FUT2 encode alpha 1, 2-fucosyltransferases which catalyze the addition of alpha 1, 2-linked fucose to glycans. Glycan products of FUT1 and FUT2, such as Globo H and Lewis Y, are highly expressed on malignant tissues, including breast cancer. Herein, we investigated the roles of FUT1 and FUT2 in breast cancer. Silencing of FUT1 or FUT2 by shRNAs inhibited cell proliferation in vitro and tumorigenicity in mice. This was associated with diminished properties of cancer stem cell (CSC), including mammosphere formation and CSC marker both in vitro and in xenografts. Silencing of FUT2, but not FUT1, significantly changed the cuboidal morphology to dense clusters of small and round cells with reduced adhesion to polystyrene and extracellular matrix, including laminin, fibronectin and collagen. Silencing of FUT1 or FUT2 suppressed cell migration in wound healing assay, whereas FUT1 and FUT2 overexpression increased cell migration and invasion in vitro and metastasis of breast cancer in vivo. A decrease in mesenchymal like markers such as fibronectin, vimentin, and twist, along with increased epithelial like marker, E-cadherin, was observed upon FUT1/2 knockdown, while the opposite was noted by overexpression of FUT1 or FUT2. As expected, FUT1 or FUT2 knockdown reduced Globo H, whereas FUT1 or FUT2 overexpression showed contrary effects. Exogenous addition of Globo H-ceramide reversed the suppression of cell migration by FUT1 knockdown but not the inhibition of cell adhesion by FUT2 silencing, suggesting that at least part of the effects of FUT1/2 knockdown were mediated by Globo H. Our results imply that FUT1 and FUT2 play important roles in regulating growth, adhesion, migration and CSC properties of breast cancer, and may serve as therapeutic targets for breast cancer

Validation of novel biomarkers for colorectal cancer detection and production of novel antibodies against E-selectin ligands

In 2018, colorectal cancer (CRC) remains the second deadliest kind of cancer with 881,00 deaths of the 1.8 million new cases. Late stages detection is more likely to develop recurrences, even after treatment, leading to the necessity to create a new system to early stages detection. Thus, understanding the biology of the cancer and biomarker discovery are important steps in cancer research. Several studies on cancer-associated glycosylation revealed that aberrant glycosylation is a universal feature in various steps of malignant transformation and tumour progression. Aberrant glycosylation is associated with poor survival, cancer progression, and metastasis, such as overexpression of Thomsen-nouvelle (Tn)-, T-, and sialyl-T (sT) antigens. Main carriers of sT- and sTn-antigens were identified as the mucin MUC1 and CD44v6. On the other hand, the Lewis antigens and their sialylated derives (Lex/sLex and Lea/sLeA) are the most prominent cancer-associated epitopes on both glycoproteins and glycolipids, since their overexpression is related to CRC malignant transformations and may lead to increased tumour cell adhesion and motility, thereby resulting in metastasis. The project aims to discover new potential biomarkers for CRC early detection and to evaluate the therapeutic potential of antibodies against CRC-associated antigens, namely against Lewis antigens and Thomsen-nouvelle (Tn)-, T-, and sialyl-T (sT) antigens. For the same reason glycoproteins, such as MUC1, CAE and CD44 will be studied for the development of novel monoclonal antibody production. After biomarker validation, hybridoma technology has been chosen to produce novel antibodies against, sLe and/or CD44, immunizing the mice directly with cancer cell lines proteins. The hybridoma against CD44 show staining against CD44 and sLex/a and further screening must be performed. The second hybridoma line against sLe antigens also shows positivity against different total cell lysate of CRC cells. Two clones have been selected for further characterization. The clones will be validated either for CRC early diagnosis or CRC treatment potential