Background: Aberrant protein glycosylation is a common feature of cancer and contributes to malignant behavior. However, how and to what extent the cellular glycome is involved in cancer development and progression is still undefined. The primary objective of this study is to conduct insilico identification of glycome genes that could reveal a signature of cancer using expression profiles of cancer genomes. There exists a list of ~500 glycome genes in several molecular categories. This study is based on the hypothesis that if the glycosylation is a common feature of cancer, there exists a shortlist of cancer glycome genes and their expression profiles should carry the signature capable of differentiating 33 different cancers available in The Cancer Genome Atlas (TCGA). Method: The distribution of cancer samples in TCGA is highly imbalanced, ranging from 36 for Cholangiocarcinoma (CHOL) to 1089 for Breast Cancer (BRCA). Supervised feature selection approaches to identify the signature genes would be biased to larger groups. We developed a computational framework using concrete autoencoder (CAE), a deep learning-based unsupervised feature selection algorithm, to find the cancer-related glycome genes. The criteria of optimal feature subset used in this study are (a) the number of features should be as few as possible, and (b) accuracy of classification using the selected features should be > 90%. Results: Our experiment showed a shortlist of glycome genes (132 genes) that can differentiate 33 different cancers with an accuracy of 92%. This study reflects that the cancer glycome genes signify the origins of cancer.
Galectin-1 (Gal-1), a β-galactoside-binding protein, can alter fate and effector function of Th cells; however, little is known about how Gal-1 induces Th cell differentiation. In this article, we show that both uncommitted and polarized Th cells bound by Gal-1 expressed an immunoregulatory signature defined by IL-10. IL-10 synthesis was stimulated by direct Gal-1 engagement to cell surface glycoproteins, principally CD45, on activated Th cells and enhanced by IL-21 expression through the c-Maf/aryl hydrocarbon receptor pathway, independent of APCs. Gal-1-induced IL-10(+) T cells efficiently suppressed T cell proliferation and T cell-mediated inflammation and promoted the establishment of cancer immune-privileged sites. Collectively, these findings show how Gal-1 functions as a major glycome determinant regulating Th cell development, inflammation, and tumor immunity.
E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin–dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin–dependent adhesive interactions that direct homing of human HPC to BM.
Abstract Metastatic melanoma is a lethal disease with a dismal 5-year survival rate. Thus, intense efforts to boost novel therapeutic strategies are underway to identify early detection of melanomas with a high propensity to metastasize. We recently discovered that the loss of cell surface glycan, I-antigen, corresponds with the transition of primary melanoma to metastatic melanoma. I-antigen or I-branched glycans are synthesized by β16, N-acetylglucosaminyltransferase 2 (GCNT2) and inversely correlate with the growth and signaling potential of metastatic melanoma cells. Moreover, compared with high GCNT2 expression in normal melanocytes, nevi, and early-stage primary melanomas, GCNT2 is conspicuously lost in metastatic melanomas. We anticipate the potential utilization of GCNT2 expression as a biomarker to predict melanoma metastasis. Further, metastasis and aggressive disease progression are key phenotypes of tumor-initiating cells (TIC), which are preferentially generated in areas of hypoxia. In the vertical growth phase of primary melanomas and melanoma metastases, the tumor microenvironment is typically hypoxic (1.5% oxygen). We hypothesize that the hypoxic microenvironment aids in metastatic melanoma progression through TIC generation and immune evasion, by downregulating GCNT2 and switching I-branched glycans to linear glycans. In this study, metastatic melanoma cells grown under hypoxic conditions had reduced GCNT2 and MITF with upregulated stem cell marker KLF4 expression. Importantly, in the in vivo TIC assay, we found significantly decreased tumor formation with increased GCNT2 expression while low GCNT2 levels enabled tumor formation even when 103 cells were injected in immunocompromised mice. Since TICs are thought to evade immune clearance, we investigated whether loss of GCNT2 increased TIC characteristics and also enabled immunosuppressive features. In human PBMC - metastatic melanoma co-cultures, there was an increase in T regulatory cell generation associated with low GCNT2 compared to high GCNT2 expression in melanoma cells, suggesting that loss of GCNT2 associates with increased TIC generation, tumor formation, and immunoevasion potential. Using melanoma patient specimens, immunohistochemical analysis of GCNT2 corresponded with a significant increase in mortality with the loss of GCNT2 staining. Altogether, these findings highlight GCNT2/I-branching not only as a biomarker of melanoma virulence but reveal malignancy-associated pathways functioning in parallel with loss of GCNT2/I-branching that could offer additional targets for the treatment of metastatic melanoma. Citation Format: Asmi Chakraborty, Mariana Perez, Norhan B. B Mohammed, Michael Wells, James S. Wilmott, John F. Thompson, Stuart M. Haslam, Wei Wang, Richard A. Scolyer, George F. Murphy, Charles J. Dimitroff. Hypoxia-mediated downregulation of GCNT2/I-antigen in metastatic melanoma accelerates disease progression and mortality [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 2609.
Abstract Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.
Advanced prostate cancer commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. Prostate cancer cells roll on E-selectin(+) BMEC through E-selectin ligand-binding interactions under shear flow, and prostate cancer cells exhibit firm adhesion to BMEC via β1, β4, and αVβ3 integrins in static assays. However, whether these discrete prostate cancer cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Here, we describe how metastatic prostate cancer cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. Prostate cancer cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and β1 and αVβ3 integrins. Expression analysis in human metastatic prostate cancer tissue revealed that β1 was markedly upregulated compared with expression of other β subunits. Prostate cancer cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as β1, αVβ3, Rac1, and Rap1 were constitutively active. In homing studies, prostate cancer cell trafficking to murine femurs was dependent on E-selectin ligand, β1 integrin, and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases in transgenic adenoma of mouse prostate mice dramatically reduced prostate cancer incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, β1 and αVβ3 integrins, and Rac/Rap1 GTPases in mediating prostate cancer cell homing and entry into bone and offer new insight into the role of α1,3 fucosylation in prostate cancer development.
Inflammation and cancer metastasis are associated with extravasation of leukocytes or tumor cells from blood into tissue. Such movement is believed to follow a coordinated and sequential molecular cascade initiated, in part, by the three members of the selectin family of carbohydrate-binding proteins: E-selectin (CD62E), L-selectin (CD62L) and P-selectin (CD62P). E-selectin is particularly noteworthy in disease by virtue of its expression on activated endothelium and on bone–skin microvascular linings and for its role in cell rolling, cell signaling and chemotaxis. E-selectin, along with L- or P-selectin, mediates cell tethering and rolling interactions through the recognition of sialo-fucosylated Lewis carbohydrates expressed on structurally diverse protein–lipid ligands on circulating leukocytes or tumor cells. Major advances in understanding the role of E-selectin in inflammation and cancer have been advanced by experiments assaying E-selectin-mediated rolling of leukocytes and tumor cells under hydrodynamic shear flow, by clinical models of E-selectin-dependent inflammation, by mice deficient in E-selectin and by mice deficient in glycosyltransferases that regulate the binding activity of E-selectin ligands. Here, the authors elaborate on how E-selectin and its ligands may facilitate leukocyte or tumor cell recruitment in inflammatory and metastatic settings. Antagonists that target cellular interactions with E-selectin and other members of the selectin family, including neutralizing monoclonal antibodies, competitive ligand inhibitors or metabolic carbohydrate mimetics, exemplify a growing arsenal of potentially effective therapeutics in controlling inflammation and the metastatic behavior of cancer.
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