Asmi Chakraborty’s research while affiliated with FIU Herbert Wertheim College of Medicine and other places

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TIC) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines (poly-LacNAc) via loss of I-branching enzyme, β1,6 N-acetylglucosaminyltransferase 2 (GCNT2). Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. Here, we explored hypoxia-dependent alterations in MM glycome-associated genes and found that GCNT2 was downregulated and a galectin (Gal)-8 – ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low GCNT2 levels correlated with poor patient outcome and patient serum samples were elevated for Gal-8. Depressed GCNT2 in MM cells upregulated TIC marker, nerve growth factor receptor (NGFR)/CD271, whereas loss of MM cell-intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-LacNAcs on N-glycans of the TIC marker and pro-metastatic molecule CD44, among other receptors and activated pro-survival factor AKT. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-LacNAc and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-LacNAcs and Gal-8 as biomarkers and therapeutic targets of MM.

The reported roles of the β-galactoside-binding lectin family, known as galectins, in disease development have been advancing at a remarkable pace. Galectins and their glycan counter-receptor ligands are now considered key functional determinants in malignant and metastatic progression, tumor immune evasion, autoimmunity, and immune homeostasis. Their influence in these processes is elicited through coordinated expression in tumor, immune and stromal cellular compartments. While analysis of galectin levels in related research efforts is routinely performed through immunoassays and RT-qPCR, detection, and identification of glycan counter-receptor ligands in their native form on the cell surface has lagged. In this report, we present methods to detect and identify glycan counter-receptor ligands to galectin (Gal)-3 and Gal-9-two galectins at the crosshairs of cancer and immunology research. As a model, we will describe (1) isolation of human B-cell subsets from fresh tonsil tissue, (2) assaying of Gal-3/-9-binding activities on human B cells, and (3) identifying Gal-3/-9 ligands on human B-cell surfaces. These methods, of course, can be implemented on any cell type to provide a cellular and molecular context capable of transmitting a galectin-mediated phenotype. Establishing a galectin-binding activity on specific counter-receptor ligand(s) can help unearth potential critical determinants capable of delivering cellular signals required for disease progression. These advances open new avenues of research investigation that result in novel therapeutic targets and approaches.

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.

Humoral immunity is reliant on efficient recruitment of circulating naïve B cells from blood into peripheral lymph nodes (LN) and timely transition of naive B cells to high affinity antibody (Ab)-producing cells. Current understanding of factor(s) coordinating B cell adhesion, activation and differentiation within LN, however, is incomplete. Prior studies on naïve B cells reveal remarkably strong binding to putative immunoregulator, galectin (Gal)-9, that attenuates BCR activation and signaling, implicating Gal-9 as a negative regulator in B cell biology. Here, we investigated Gal-9 localization in human tonsils and LNs and unearthed conspicuously high expression of Gal-9 on high endothelial and post-capillary venules. Adhesion analyses showed that Gal-9 can bridge human circulating and naïve B cells to vascular endothelial cells (EC), while decelerating transendothelial migration. Moreover, Gal-9 interactions with naïve B cells induced global transcription of gene families related to regulation of cell signaling and membrane/cytoskeletal dynamics. Signaling lymphocytic activation molecule F7 (SLAMF7) was among key immunoregulators elevated by Gal-9-binding, while SLAMF7's cytosolic adapter EAT-2, which is required for cell activation, was eliminated. Gal-9 also activated phosphorylation of pro-survival factor, ERK. Together, these data suggest that Gal-9 promotes B cell – EC interactions while delivering anergic signals to control B cell reactivity.

Cancer immunotherapy and its budding effectiveness at improving patient outcomes has revitalized our hope to fight cancer in a logical and safe manner. Immunotherapeutic approaches to reengage the immune system have largely focused on reversing immune checkpoint inhibitor pathways, which suppress the antitumor response. Although these approaches have generated much excitement, they still lack absolute success. Interestingly, newly described host-tumor sugar chains (glycosylations) and glycosylation-binding proteins (lectins) play key roles in evading the immune system to determine cancer progression. In this issue of the JCI, Nambiar et al. used patient head and neck tumors and a mouse model system to investigate the role of galactose-binding lectin 1 (Gal1) in immunotherapy resistance. The authors demonstrated that Gal1 can affect immune checkpoint inhibitor therapy by increasing immune checkpoint molecules and immunosuppressive signaling in the tumor. Notably, these results suggest that targeting a tumor's glycobiological state will improve treatment efficacy.