The universe of galectin-binding partners and their functions in health and disease.

Galectins, a family of evolutionarily conserved glycan-binding proteins, play key roles in diverse biological processes including tissue repair, adipogenesis, immune cell homeostasis, angiogenesis, and pathogen recognition. Dysregulation of galectins and their ligands has been observed in a wide range of pathologic conditions including cancer, autoimmune inflammation, infection, fibrosis, and metabolic disorders. Through protein-glycan or protein-protein interactions, these endogenous lectins can shape the initiation, perpetuation, and resolution of these processes, suggesting their potential roles in disease monitoring and treatment. However, despite considerable progress, a full understanding of the biology and therapeutic potential of galectins has not been reached due to their diversity, multiplicity of cell targets, and receptor promiscuity. In this article, we discuss the multiple galectin-binding partners present in different cell types, focusing on their contributions to selected physiologic and pathologic settings. Understanding the molecular bases of galectin-ligand interactions, particularly their glycan-dependency, the biochemical nature of selected receptors, and underlying signaling events, might contribute to designing rational therapeutic strategies to control a broad range of pathologic conditions.

Galectin-1 confers resistance to doxorubicin in hepatocellular carcinoma cells through modulation of P-glycoprotein expression.

Galectin-1 (GAL1), a ß-galactoside-binding protein abundantly expressed in the tumor microenvironment, has emerged as a key mechanism of chemoresistance developed by different tumors. Although increased expression of GAL1 is a hallmark of hepatocellular carcinoma (HCC) progression, aggressiveness and metastasis, limited information is available on the role of this endogenous lectin in HCC resistance to chemotherapy. Moreover, the precise mechanisms underlying this effect are uncertain. HCC has evolved different mechanisms of resistance to chemotherapy including those involving the P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, which controls intracellular drug concentration. Here, we investigated the molecular mechanism underlying GAL1-mediated chemoresistance in HCC cells, particularly the involvement of P-gp in this effect. Our results show that GAL1 protected HepG2 cells from doxorubicin (DOX)- and sorafenib-induced cell death in vitro. Accordingly, GAL1-overexpressing HepG2 cells generated DOX-resistant tumors in vivo. High expression of GAL1 in HepG2 cells reduced intracellular accumulation of DOX likely by increasing P-gp protein expression rather than altering its membrane localization. GAL1-mediated increase of P-gp expression involved activation of the phosphatidylinositol-3 kinase (PI3K) signaling pathway. Moreover, 'loss-of-function' experiments revealed that P-gp mediates GAL1-driven resistance to DOX, but not to sorafenib, in HepG2 cells. Conversely, in PLC/PRF/5 cells, P-gp protein expression was undetectable and GAL1 did not control resistance to DOX or sorafenib, supporting the critical role of P-gp in mediating GAL1 effects. Collectively, our findings suggest that GAL1 confers chemoresistance in HCC through mechanisms involving modulation of P-gp, thus emphasizing the role of this lectin as a potential therapeutic target in HCC.

ALCAM/CD166: A pleiotropic mediator of cell adhesion, stemness and cancer progression.

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) is a glycoprotein involved in homotypic and heterotypic cell adhesion. ALCAM can be proteolytically cleaved at the cell surface by metalloproteases, which generate shedding of its ectodomain. In various tumors, ALCAM is overexpressed and serves as a valuable prognostic marker of disease progression. Moreover, CD166 has been identified as a putative cancer stem cell marker in particular cancers. Herein, we summarize biochemical aspects of ALCAM, including structure, proteolytic shedding, alternative splicing, and specific ligands, and integrate this information with biological functions of this glycoprotein including cell adhesion, migration and invasion. In addition, we discuss different patterns of ALCAM expression in distinct tumor types and its contribution to tumor progression. Finally, we highlight the role of ALCAM as a cancer stem cell marker and introduce current clinical trials associated with this molecule. Future studies are needed to define the value of shed ALCAM in biofluids or ALCAM isoform expression as prognostic biomarkers in tumor progression.

Full-length galectin-8 and separate carbohydrate recognition domains: the whole is greater than the sum of its parts?

Galectin-8 (Gal-8) is a tandem-repeat type galectin with affinity for ß-galactosides, bearing two carbohydrate recognition domains (CRD) connected by a linker peptide. The N- and C-terminal domains (Gal-8N and Gal-8C) share 35% homology, and their glycan ligand specificity is notably dissimilar: while Gal-8N shows strong affinity for α(2-3)-sialylated oligosaccharides, Gal-8C has higher affinity for non-sialylated oligosaccharides, including poly-N-acetyllactosamine and/ or A and B blood group structures. Particularly relevant for understanding the biological role of this lectin, full-length Gal-8 can bind cell surface glycoconjugates with broader affinity than the isolated Gal-8N and Gal-8C domains, a trait also described for other tandem-repeat galectins. Herein, we aim to discuss the potential use of separate CRDs in modelling tandem-repeat galectin-8 and its biological functions. For this purpose, we will cover several aspects of the structure-function relationship of this protein including crystallographic structures, glycan specificity, cell function and biological roles, with the ultimate goal of understanding the potential role of each CRD in predicting full-length Gal-8 involvement in relevant biological processes.

Dual knockdown of Galectin-8 and its glycosylated ligand, the activated leukocyte cell adhesion molecule (ALCAM/CD166), synergistically delays in vivo breast cancer growth.

Galectin-8 (Gal-8), a 'tandem-repeat'-type galectin, has been described as a modulator of cellular functions including adhesion, spreading, growth arrest, apoptosis, pathogen recognition, autophagy, and immunomodulation. We have previously shown that activated leukocyte cell adhesion molecule (ALCAM), also known as CD166, serves as a receptor for endogenous Gal-8. ALCAM is a member of the immunoglobulin superfamily involved in cell-cell adhesion through homophilic (ALCAM-ALCAM) and heterophilic (i.e. ALCAM-CD6) interactions in different tissues. Here we investigated the physiologic relevance of ALCAM-Gal-8 association and glycosylation-dependent mechanisms governing these interactions. We found that silencing of ALCAM in MDA-MB-231 triple negative breast cancer cells decreases cell adhesion and migration onto Gal-8-coated surfaces in a glycan-dependent fashion. Remarkably, either Gal-8 or ALCAM silencing also disrupted cell-cell adhesion, and led to reduced tumor growth in a murine model of triple negative breast cancer. Moreover, structural characterization of endogenous ALCAM N-glycosylation showed abundant permissive structures for Gal-8 binding. Importantly, we also found that cell sialylation controls Gal-8-mediated cell adhesion. Altogether, these findings demonstrate a central role of either ALCAM or Gal-8 (or both) in controlling triple negative breast cancer.

Galectins: Multitask signaling molecules linking fibroblast, endothelial and immune cell programs in the tumor microenvironment.

Tumor cells corrupt surrounding normal cells instructing them to support proliferative, pro-angiogenic and immunosuppressive networks that favor tumorigenesis and metastasis. This dynamic cross-talk is sustained by a range of intracellular signals and extracellular mediators produced by both tumoral and non-tumoral cells. Galectins -whether secreted or intracellularly expressed- play central roles in the tumorigenic process by delivering regulatory signals that contribute to reprogram fibroblasts, endothelial and immune cell programs. Through glycosylation-dependent or independent mechanisms, these endogenous lectins control a variety of cellular events leading to tumor cell proliferation, survival, migration, inflammation, angiogenesis and immune escape. Here we discuss the role of galectin-driven pathways, particularly those activated in non-tumoral stromal cells, in modulating tumor progression.

Glycosylation-dependent binding of galectin-8 to activated leukocyte cell adhesion molecule (ALCAM/CD166) promotes its surface segregation on breast cancer cells.

BACKGROUND: We previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell-cell interactions. Gal-8 is a "tandem-repeat"-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon. METHODS: We performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization. RESULTS: We showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells. CONCLUSIONS: Our data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms. GENERAL SIGNIFICANCE: A novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.

Galectin-1 Controls the Proliferation and Migration of Liver Sinusoidal Endothelial Cells and Their Interaction With Hepatocarcinoma Cells.

Galectin-1 (Gal1), a ß-galactoside-binding protein elevated in hepatocellular carcinoma (HCC), promotes epithelial-mesenchymal transition (EMT) and its expression correlates with HCC growth, invasiveness, and metastasis. During the early stages of HCC, transforming growth factor ß1 (TGF-ß1 ) acts as a tumor suppressor; however in advanced stages, HCC cells lose their cytostatic response to TGF-ß1 and undergo EMT. Here, we investigated the role of Gal1 on liver endothelial cell biology, and the interplay between Gal1 and TGF-ß1 in HCC progression. By Western blot and immunofluorescence, we analyzed Gal1 expression, secretion and localization in HepG2 and HuH-7 human HCC cells, and in SK-HEP-1 human liver sinusoidal endothelial cells (SECs). We used loss-of-function and gain-of-function experiments to down- or up-regulate Gal1 expression, respectively, in HepG2 cells. We cultured SK-HEP-1 cells with conditioned media from HCC cells secreting different levels of Gal1, and demonstrated that Gal1 derived from tumor hepatocytes induced its own expression in SECs. Colorimetric and scratch-wound assays revealed that secretion of Gal1 by HCC cells induced SEC proliferation and migration. Moreover, by fluorescence microscopy we demonstrated that Gal1 promoted glycan-dependent heterotypic adhesion of HepG2 cells to SK-HEP-1 SECs. Furthermore, TGF-ß1 induced Gal1 expression and secretion by HCC cells, and promoted HepG2 cell adhesion to SK-HEP-1 SECs through a Gal1-dependent mechanism. Finally, Gal1 modulated HepG2 cell proliferation and sensitivity to TGF-ß1 -induced growth inhibition. Our results suggest that Gal1 and TGF-ß1 might function coordinately within the HCC microenvironment to regulate tumor growth, invasion, metastasis, and angiogenesis.

Assembly, organization and regulation of cell-surface receptors by lectin-glycan complexes.

Galectins are a family of ß-galactoside-binding lectins carrying at least one consensus sequence in the carbohydrate-recognition domain. Properties of glycosylated ligands, such as N- and O-glycan branching, LacNAc (N-acetyl-lactosamine) content and the balance of α2,3- and α2,6-linked sialic acid dramatically influence galectin binding to a preferential set of counter-receptors. The presentation of specific glycans in galectin-binding partners is also critical, as proper orientation and clustering of oligosaccharide ligands on multiple carbohydrate side chains increase the binding avidity of galectins for particular glycosylated receptors. When galectins are released from the cells, they typically concentrate on the cell surface and the local matrix, raising their local concentration. Thus galectins can form their own multimers in the extracellular milieu, which in turn cross-link glycoconjugates on the cell surface generating galectin-glycan complexes that modulate intracellular signalling pathways, thus regulating cellular processes such as apoptosis, proliferation, migration and angiogenesis. Subtle changes in receptor expression, rates of protein synthesis, activities of Golgi enzymes, metabolite concentrations supporting glycan biosynthesis, density of glycans, strength of protein-protein interactions at the plasma membrane and stoichiometry may modify galectin-glycan complexes. Although galectins are key contributors to the formation of these extended glycan complexes leading to promotion of receptor segregation/clustering, and inhibition of receptor internalization by surface retention, when these complexes are disrupted, some galectins, particularly galectin-3 and -4, showed the ability to drive clathrin-independent mechanisms of endocytosis. In the present review, we summarize the data available on the assembly, hierarchical organization and regulation of conspicuous galectin-glycan complexes, and their implications in health and disease.

Galectin-8: a matricellular lectin with key roles in angiogenesis.

Galectin-8 (gal-8) is a "tandem-repeat"-type galectin, containing two carbohydrate recognition domains connected by a linker peptide. gal-8 is expressed both in the cytoplasm and nucleus in vascular endothelial cells (ECs) from normal and tumor-associated blood vessels, and in lymphatic endothelial cells. Herein, we describe a novel role for gal-8 in the regulation of vascular and lymphatic angiogenesis and provide evidence of its critical implications in tumor biology. Functional assays revealed central roles for gal-8 in the control of capillary-tube formation, EC migration and in vivo angiogenesis. So far, two endothelial ligands have been described for gal-8, namely podoplanin in lymphatic vessels and CD166 (ALCAM, activated leukocyte cell adhesion molecule) in vascular ECs. Other related gal-8 functions are also summarized here, including cell adhesion and migration, which collectively demonstrate the multi-functionality of this complex lectin. Thus, gal-8 is an important component of the angiogenesis network, and an essential molecule in the extracellular matrix by providing molecular anchoring to this surrounding matrix. The implications of gal-8 in tumor angiogenesis remain to be further explored, but it is exciting to speculate that modulating gal-8-glycan interactions could be used to block lymphatic-vascular connections vital for metastasis.