Galectin-3 Binding Research Articles

Sticking to Sugars at the Metastatic Site: Sialyltransferase ST6GalNAc2 Acts as a Breast Cancer Metastasis Suppressor

O-linked glycans on plasma membrane proteins are altered in cancer cells, leading to changes in cell adhesive properties and contributing to metastasis. Mechanisms of how these carbohydrates alter tumor spread remain vague. In this issue of Cancer Discovery, Murugaesu and colleagues, using an in vivo functional RNA interference metastasis screen, identified sialyltransferase ST6GalNAc2 as a novel metastasis suppressor gene. Aggressive estrogen receptor-negative breast cancers with reduced ST6GalNAc2 expression alter O-linked glycans on tumor cell surface, increasing soluble lectin galectin-3 binding and tumor cell clustering at metastatic sites.

Glycan-dependent binding of galectin-1 to neuropilin-1 promotes axonal regeneration after spinal cord injury

Following spinal cord injury (SCI), semaphorin 3A (Sema3A) prevents axonal regeneration through binding to the neuropilin-1 (NRP-1)/PlexinA4 receptor complex. Here, we show that galectin-1 (Gal-1), an endogenous glycan-binding protein, selectively bound to the NRP-1/PlexinA4 receptor complex in injured neurons through a glycan-dependent mechanism, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. Although both Gal-1 and its monomeric variant contribute to de-activation of microglia, only high concentrations of wild-type Gal-1 (which co-exists in a monomer-dimer equilibrium) bind to the NRP-1/PlexinA4 receptor complex and promote axonal regeneration. Our results show that Gal-1, mainly in its dimeric form, promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A binding to NRP-1/PlexinA4 complex, supporting the use of this lectin for the treatment of SCI patients.

Glycosylation-Dependent Lectin-Receptor Interactions Preserve Angiogenesis in Anti-VEGF Refractory Tumors

The clinical benefit conferred by vascular endothelial growth factors (VEGF)-targeted therapies is variable, and tumors from treated patients eventually reinitiate growth. Here, we identify a glycosylation-dependent pathway that compensates for the absence of cognate ligand and preserves angiogenesis in response to VEGF blockade. Remodeling of the endothelial cell (EC) surface glycome selectively regulated binding of galectin-1 (Gal1), which upon recognition of complex N-glycans on VEGFR2, activated VEGF-like signaling. Vessels within anti-VEGF-sensitive tumors exhibited high levels of α2-6-linked sialic acid, which prevented Gal1 binding. In contrast, anti-VEGF refractory tumors secreted increased Gal1 and their associated vasculature displayed glycosylation patterns that facilitated Gal1-EC interactions. Interruption of β1-6GlcNAc branching in ECs or silencing of tumor-derived Gal1 converted refractory into anti-VEGF-sensitive tumors, whereas elimination of α2-6-linked sialic acid conferred resistance to anti-VEGF. Disruption of the Gal1-N-glycan axis promoted vascular remodeling, immune cell influx and tumor growth inhibition. Thus, targeting glycosylation-dependent lectin-receptor interactions may increase the efficacy of anti-VEGF treatment.

Synthesis of 1,2,3-triazole-linked galactohybrids and their inhibitory activities on galectins

Here a synthesis of novel galactose-1,2,3-triazole conjugates is described. The title compounds were obtained from 3-azido-3-deoxy-1,2: 5,6-di-O-isopropylidene-alpha-D-galactofuranose via a copper catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction. It was demonstrated that the title compounds in their isopropylidene-protected form tend to chelate copper. The copper content can be diminished to 10 ppm by successive treatment with EDTA and Na2S followed by chromatographic purification. Acidic hydrolysis of the acetonide protecting groups provided water soluble galactohybrids that were tested for their affinity towards galectin-1 and galectin-3. The trimeric galactohybrid exhibited a 160-fold preference for galectin-3 binding with K-d 50 mu M. One of the obtained disaccharides was characterized by X-ray analysis. (Less)

Galectin-3 Enhances Epithelial Cell Adhesion and Wound Healing in Rat Cornea

Background/Aims: To investigate if galectin-3: (1) enhances adhesion of rat corneal epithelial cells onto a collagen IV substrate and (2) promotes wound healing in rat corneal explants. Methods: Primary cultures of rat corneal epithelial cells were fixed and immunostained with galectin-3 antibody. To test cellular adherence onto plates coated with collagen type IV, isolated corneal epithelial cells from rats were cultured for 24 h with or without recombinant galectin-3. The attached cells were counted after fixing and staining with 0.1% crystal violet. Direct binding of galectin-3 to collagen IV was tested using a biotin label transfer method. To evaluate wound healing, explants with a 3.5-mm diameter wound in the central corneal epithelium from rats were incubated for 16 h with or without recombinant galectin-3. Changes in the size of the wound were measured with a digital microscope after staining with 5% fluorescein sodium. Results: In rat corneal epithelial cells, galectin-3 was stained throughout the cytoplasm, with increasing density adjacent to the plasma membrane. Exogenous galectin-3, but not epidermal growth factor (EGF), significantly promoted adhesion of corneal epithelial cells onto the collagen IV substrate. Galectin-3 directly bound to collagen IV in vitro. Exogenous galectin-3 significantly enhances wound healing in the corneal explants, which was partially inhibited by β-lactose. Conclusion: Galectin-3 promotes adhesion of corneal epithelial cells onto collagen IV and enhances wound healing in corneal explants. Since galectin-3 functions in promoting wound healing by a different mechanism than that used by EGF, exogenous galectin-3 may be a candidate drug for enhancing epithelial cell wound healing in disorders of the cornea.

Ligand binding and complex formation of galectin-3 is modulated by pH variations

Galectin-3-dependent clusters or lattices are formed at the surface as well as in distinct organelles of eukaryotic cells. Incorporation into membrane proximal networks can fix glycoproteins within subcellular domains or sort them into distinct transport pathways. In the present paper we analysed the effect of acidification on the sugar binding and self-oligomerization of galectin-3. Using a fluorescence anisotropy assay we measured decreasing galectin-3 affinities to the blood group antigen GalNAcα1-3(Fucα1-2)Galβ1-4Glc under low pH conditions. Binding to the strong interaction partner N-acetyl-D-lactosamine was also lost at pH 5.0, whereas the less efficient ligand lactose was still able to bind. This indicates that variations in the binding specificity to distinct glycans can be observed by altering the pH. The formation of galectin-3-based complexes by interaction with the multivalent glycoproteins asialofetuin or transferrin was also obliterated at acidic pH and the ligand-binding affinity itself was modulated by oligomerization of the lectin. When galectin-3 was added to giant plasma membrane vesicles from the apical surface of epithelial cells, pH modulation could generate or eliminate the formation of membrane domains enriched with p75(NTR) (neurotrophin receptor p75). In conclusion, the results of the present study suggest that the formation and composition of galectin-3 networks can be fine-tuned by changes in the environmental pH.

Human Milk Galectin-3 Binding Protein and Breast-feeding–associated HIV Transmission

Analysis of milk from 247 HIV-infected Zambian mothers showed that galectin-3 binding protein concentrations were significantly higher among HIV-infected mothers who transmitted HIV through breast-feeding (6.51 ± 2.12 μg/mL) than among nontransmitters but were also correlated with higher milk and plasma HIV RNA copies/mL and lower CD4+ cell counts. The association between galectin-3 binding protein and postnatal transmission was attenuated after adjustment for milk and plasma HIV load and CD4+ cell counts. This suggests that although milk galectin-3 binding protein is a marker of advanced maternal disease, it does not independently modify transmission risk.

Identification Of Galectin-1 and Galectin-3 As Novel Binding Partners For Factor VIII

During biosynthesis, Factor VIII (FVIII) undergoes complex post-translational modification including significant glycosylation. Consequently each FVIII molecule can contains 25 N- and 6 O-linked glycans. These carbohydrate structures are of physiological significance. For example, FVIII glycan expression modulates intracellular trafficking and also regulates FVIII clearance by dendritic cells. Nevertheless, the molecular mechanisms through which glycan structures influence FVIII biology remains poorly defined. Interestingly, carbohydrate-binding galectins (Gal) -1 and -3 have recently been reported to bind human VWF. Moreover, these galectin interactions significantly influence VWF function. In this study, based upon similar glycans expression profiles, we hypothesised that galectins might also constitute novel binding partners for human FVIII.In brief, His-tagged Gal-1 and Gal-3 were expressed in E-coli and purified using nickel chromatography. Recombinant FVIII (rFVIII) was purified from different commercial concentrates. Subsequently, FVIII glycosylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. Galectin-FVIII interaction was characterised using modified immunosorbant assays and surface plasmon resonance (SPR).In plate–binding assays using purified proteins and SPR studies, both Gal-1 and Gal-3 bound to full length rFVIII in a time- and dose-dependent manner. Interestingly the apparent affinities of the galectin-FVIII interactions (Kd of 0.11 ± 0.02nM for Gal-1 and 0.21 ± 0.1nM for Gal-3 respectively) were unusually high for these lectins. Digestion with PNGase F to remove N-linked glycans ablated FVIII binding to Gal-1 (8.6 ± 1%; p<0.0001). In contrast, PNG-FVIII retained significant ability to bind Gal-3 (30.3 ± 3%; p<0.0001). However, combined FVIII digestion with both PNGase F and O glycosidase further attenuated Gal-3 binding (16.5 ± 2%; p<0.05). Cumulatively these findings suggest that whilst Gal-1 binding is mediated predominantly through the N-linked glycans of FVIII, both N- and O-linked glycans modulate its interaction with Gal-3.The majority of FVIII glycans are contained within the B domain. Unsurprisingly, Gal-1 and Gal-3 binding were both markedly attenuated for B domain deleted rFVIII compared to full length rFVIII (42 ± 1% and 26 ± 0.8%; p<0.0001). Previous studies have described different glycosylation profiles for specific full length commercial rFVIII products. To investigate the relevance of this differential glycosylation, we compared the galectin-binding properties of Advate® (CHO cell line) and Helixate® (BHK cell line). Interestingly, Gal-1 and Gal-3 both displayed significantly enhanced affinity for Helixate (107 ± 2% and 124 ± 1%; p<0.05). These findings are consistent with the fact that the N-linked glycans of BHK-derived FVIII express galactose α1-3 galactose epitopes which constitute preferential galectin-binding ligands.To determine whether FVIII interacts with galectins in vivo, immunoprecipitation studies were performed using plasma from VWF-/- mice. We observed that that both Gal-1 and Gal-3 were co-precipitated with FVIII even in the absence of VWF. Consequently, both the VWF-FVIII complex and free FVIII in plasma are likely to circulate in a complex with galectins. Importantly, recent studies have reported a prothrombotic phenotype in Gal-1/Gal-3 double deficient mice compared to wild type controls following ferric chloride injury. To investigate whether galectin-binding influences FVIII function, FVIII activity was assessed using a one-stage clotting assay in the presence of increasing galectin concentrations. Interestingly, preincubation of FVIII with Gal-1 (0.5-17µM) resulted in a significant dose-dependent prolongation of the APTT (58 ± 0.2 sec compared to 26 ± 3 secs, p<0.001) In contrast, no such effect was observed for galectin-3 up to 20µM, suggesting these galectins may have differential effects on FVIII biology.In conclusion, we identify Gal-1 and Gal-3 as novel direct ligands for human FVIII. Both the N- and O-linked carbohydrates of FVIII contribute to galectin binding. Importantly, different commercial FVIII concentrates do not interact with galectins in the same manner. Finally, we also demonstrate that plasma FVIII can circulate in complex with both Gal-1 and Gal-3, and that Gal-1 binding can inhibit the procoagulant function of FVIII. Disclosures:No relevant conflicts of interest to declare.

The Inhibitory Effects of a Rhamnogalacturonan Ι (RG-I) Domain from Ginseng Pectin on Galectin-3 and Its Structure-Activity Relationship

Pectin has been shown to inhibit the actions of galectin-3, a β-galactoside-binding protein associated with cancer progression. The structural features of pectin involved in this activity remain unclear. We investigated the effects of different ginseng pectins on galectin-3 action. The rhamnogalacturonan I-rich pectin fragment, RG-I-4, potently inhibited galectin-3-mediated hemagglutination, cancer cell adhesion and homotypic aggregation, and binding of galectin-3 to T-cells. RG-I-4 specifically bound to the carbohydrate recognition domain of galectin-3 with a dissociation constant of 22.2 nm, which was determined by surface plasmon resonance analysis. The structure-activity relationship of RG-I-4 was investigated by modifying the structure through various enzymatic and chemical methods followed by activity tests. The results showed that (a) galactan side chains were essential to the activity of RG-I-4, whereas arabinan side chains positively or negatively regulated the activity depending on their location within the RG-I-4 molecule. (b) The activity of galactan chain was proportional to its length up to 4 Gal residues and largely unchanged thereafter. (c) The majority of galactan side chains in RG-I-4 were short with low activities. (d) The high activity of RG-I-4 resulted from the cooperative action of these side chains. (e) The backbone of the molecule was very important to RG-I-4 activity, possibly by maintaining a structural conformation of the whole molecule. (f) The isolated backbone could bind galectin-3, which was insensitive to lactose treatment. The novel discovery that the side chains and backbone play distinct roles in regulating RG-I-4 activity is valuable for producing highly active pectin-based galectin-3 inhibitors.

Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer

Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3(-/-)/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3(-/-)/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ∼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer.

Galectin-3 Guides Intracellular Trafficking of Some Human Serotransferrin Glycoforms

Transferrin internalization via clathrin-mediated endocytosis and subsequent recycling after iron delivery has been extensively studied. Here we demonstrate a previously unrecognized parameter regulating this recycling, the binding of galectin-3 to particular glycoforms of transferrin. Two fractions of transferrin, separated by affinity chromatography based on their binding or not to galectin-3, are targeted to kinetically different endocytic pathways in HFL-1 cells expressing galectin-3 but not in SKBR3 cells lacking galectin-3; the SKBR3 cells, however, can acquire the ability to target these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3-bound glycoform increased in cancer, suggesting a pathophysiological regulation. These are novel aspects of transferrin cell biology, which has previously considered only a degree of iron loading, but not other forms of heterogeneity.

Modulation of Ocular Surface Glycocalyx Barrier Function by a Galectin-3 N-terminal Deletion Mutant and Membrane-Anchored Synthetic Glycopolymers

BackgroundInteraction of transmembrane mucins with the multivalent carbohydrate-binding protein galectin-3 is critical to maintaining the integrity of the ocular surface epithelial glycocalyx. This study aimed to determine whether disruption of galectin-3 multimerization and insertion of synthetic glycopolymers in the plasma membrane could be used to modulate glycocalyx barrier function in corneal epithelial cells.Methodology/Principal FindingsAbrogation of galectin-3 biosynthesis in multilayered cultures of human corneal epithelial cells using siRNA, and in galectin-3 null mice, resulted in significant loss of corneal barrier function, as indicated by increased permeability to the rose bengal diagnostic dye. Addition of β-lactose, a competitive carbohydrate inhibitor of galectin-3 binding activity, to the cell culture system, transiently disrupted barrier function. In these experiments, treatment with a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, but not full-length galectin-3, prevented the recovery of barrier function to basal levels. As determined by fluorescence microscopy, both cellobiose- and lactose-containing glycopolymers incorporated into apical membranes of corneal epithelial cells, independently of the chain length distribution of the densely glycosylated, polymeric backbones. Membrane incorporation of cellobiose glycopolymers impaired barrier function in corneal epithelial cells, contrary to their lactose-containing counterparts, which bound to galectin-3 in pull-down assays.Conclusions/SignificanceThese results indicate that galectin-3 multimerization and surface recognition of lactosyl residues is required to maintain glycocalyx barrier function at the ocular surface. Transient modification of galectin-3 binding could be therapeutically used to enhance the efficiency of topical drug delivery.

Identification of candidate biomarkers for hepatocellular carcinoma in plasma of HCV‐infected cirrhotic patients by 2‐D DIGE

The current methods available for screening and detecting hepatocellular carcinoma (HCC) have insufficient sensitivity and specificity, and only a low percentage of diagnosis of small tumours is based on these assays. Because HCC is usually asymptomatic at potentially curative stages, identification of biomarkers for the early detection of HCC is essential to improve patient survival. The aim of this study was to identify candidate markers for HCC development in the plasma from hepatitis C virus (HCV)-infected cirrhotic patients. We compared protein expression profiles of plasma samples from HCV-infected cirrhotic patients with and without HCC, using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) coupled with MALDI-TOF/TOF mass spectrometry. The 2-D DIGE results were analysed statistically using Decyder™ software, and verified by western blot and enzyme-linked immunosorbent assay (ELISA). In the plasma of HCV-infected HCC patients, we observed decreased expression of complement component 9, ficolin-3 (FCN3), serum amyloid P component (SAP), fibrinogen-gamma and immunoglobulin gamma-1 chain, and increased expression of vitronectin (VTN) and galectin-3 binding protein (G3BP) by DIGE analysis. ELISA confirmed DIGE results for VTN and G3BP but not for SAP or FCN3 in a larger patient population. The proteins VTN and SAP are candidate biomarkers for HCC development in HCV-infected cirrhotic patients.

Serum proteomics in amnestic mild cognitive impairment

We have explored proteins related to mild cognitive impairment (MCI). The serum proteome of 35 amnestic MCI patients and 35 cognitively healthy persons was investigated by LC MS. We identified 108 differentially expressed peptides between MCI patients and controls, belonging to 39 proteins. Eight proteins were selected for further investigation by quantitative protein measurements using a MRM assay; apolipoprotein E, carboxypeptidase N subunit 2, complement factor B (CFAB), galectin-3 binding protein (LG3BP), lumican, serum amyloid A-4 protein (SAA4), serum amyloid P-component, and sex hormone binding globulin. Results of the quantitative protein measurements showed significantly decreased levels of carboxypeptidase N subunit 2, CFAB, LG3BP, SAA4, and serum amyloid P-component in serum from amnestic MCI patients compared with cognitive healthy controls (two-sided t-test; p < 0.05). Apolipoprotein E and lumican showed no significant difference in protein levels, sex hormone binding globulin could not be quantified since the MRM assay did not reach the required sensitivity. A model based on the three most significantly decreased proteins (CFAB, LG3BP, and SAA4) showed a sensitivity and specificity of 73 and 66%, respectively, for the initial sample set. A small external validation set yielded 77% sensitivity and 75% specificity.

Investigation into the Feasibility of Thioditaloside as a Novel Scaffold for Galectin‐3‐Specific Inhibitors

Galectin-3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this β-galactose-binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin-1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour-challenged mice. A novel approach--replacing galactose with its C2 epimer, talose--offers an alternative framework, as extensions at C2 permit exploitation of a galectin-3-specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin-3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin-3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.

Proteomic identification of galectin-3 binding ligands and characterization of galectin-3 proteolytic cleavage in human prostasomes

Galectin-3 is a multifunctional carbohydrate-binding protein that was previously characterized as a proteolytic substrate for prostate-specific antigen (PSA) and was shown to be associated with prostasomes in human semen. Prostasomes are exosome-like vesicles that are secreted by the prostatic epithelium and have multiple proposed functions in normal reproduction and prostate cancer. In the current study, galectin-3 binding ligands in human prostasomes were identified and characterized with the goal to investigate galectin-3 function in prostasomes. Galectin-3 binding proteins were isolated by affinity column chromatography. Candidate ligands identified by MS/MS were PSA, prostatic acid phosphatase (PAP), zinc alpha-2-glycoprotein (ZAG), dipeptidyl peptidase-4 (CD26), aminopeptidase N (CD13), neprilysin, clusterin, antibacterial protein (FALL-39) and alpha-1-acid glycoprotein (ORM1). Biochemical methods were used to characterize the ability of galectin-3 to bind to selected ligands, and galectin-3 cleavage assays were utilized to investigate the protease(s) in prostasomes that cleaves galectin-3. CD26, CD13, PSA, PAP and ZAG immunoreactivity were detected in extracts of purified prostasomes. One-dimensional electroblot analysis of prostasomes demonstrated that CD26, PAP and CD13 immunoreactivity co-migrated with galectin-3-reactive protein bands. PSA and ZAG were found to be associated with the surface of prostasomes. Both intact and cleaved galectin-3 were detected in prostate and prostasome extracts. Cleavage and inhibition assays indicated that PSA in prostasomes proteolytically cleaves galectin-3. The identification of these glycoproteins as galectin-3 ligands lays the groundwork for future studies of galectin-3 and prostasome function in reproduction and prostate cancer.

O01.2 Innate Immunity Modulation by Trichomonas Vaginalis Galectin-Binding Glycolipid Domains

Background Trichomonas vaginalis is a protozoan extracellular parasite causing long-lasting and recurrent vaginitis with a wide range of symptoms and increased risk of HIV and other viral STIs. The protozoan...

Galectin-3– and phospho-caveolin-1–dependent outside-in integrin signaling mediates the EGF motogenic response in mammary cancer cells

In murine mammary epithelial cancer cells, galectin-3 binding to β1,6-acetylglucosaminyltransferase V (Mgat5)-modified N-glycans restricts epidermal growth factor (EGF) receptor mobility in the plasma membrane and acts synergistically with phospho-caveolin-1 to promote integrin-dependent matrix remodeling and cell migration. We show that EGF signaling to RhoA is galectin-3 and phospho-caveolin-1 dependent and promotes the formation of transient, actin-rich, circular dorsal ruffles (CDRs), cell migration, and fibronectin fibrillogenesis via Src- and integrin-linked kinase (ILK)-dependent signaling. ILK, Src, and galectin-3 also mediate EGF stimulation of caveolin-1 phosphorylation. Direct activation of integrin with Mn2+ induces galectin-3, ILK, and Src-dependent RhoA activation and caveolin-1 phosphorylation. This suggests that in response to EGF, galectin-3 enables outside-in integrin signaling stimulating phospho-caveolin-1-dependent RhoA activation, actin reorganization in CDRs, cell migration, and fibronectin remodeling. Similarly, caveolin-1/galectin-3-dependent EGF signaling induces motility, peripheral actin ruffling, and RhoA activation in MDA-MB-231 human breast carcinoma cells, but not HeLa cells. These studies define a galectin-3/phospho-caveolin-1/RhoA signaling module that mediates integrin signaling downstream of growth factor activation, leading to actin and matrix remodeling and tumor cell migration in metastatic cancer cells.

Abstract 1263: Regulation of tumor-specific CD8 T cells via interactions between galectin-3 and surface glycoproteins.

Abstract This study is testing the hypothesis that galectin-3, a 31 KDa carbohydrate-binding lectin, is involved in regulating T cell function within the tumor micro-environment by interacting with oligosaccharide chains on cell surface glycoproteins. Our interest in galectin-3 arose when we found that patients responding to a granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic pancreatic tumor vaccine developed post immunization antibody responses to galectin-3 on a proteomic screen. Oligosaccharide chains on plasma membrane proteins form a thick coat known as the glycocalyx that surrounds the entire cell. The glycocalyx has been shown to be important in regulating a diverse array of cellular processes such as adhesion, growth, and trafficking via its interactions with ligands on neighboring cells or in the extracellular environment. Lectin mediated clustering of receptors can lead to activation of downstream signaling proteins or even impact the half-life of a receptor on the cell-surface, thus affecting the duration of signaling. Changes in oligosaccharide structure through differential expression of enzymes involved in the glycosylation pathway may therefore modulate cellular activity by altering the availability and structure of binding motifs for glycan-binding proteins. Thus, the importance of lectin-glycan interactions in tumor development and the corresponding anti-tumor immune response cannot be ignored. Galectin-3 is over-expressed in several different malignancies. It has also been shown to modulate T cell responses through a diverse array of mechanisms including induction of apoptosis, TCR cross linking in CD8+ T cells, and T cell receptor (TCR) down regulation in CD4+ T cells. We have used the HER-2/neu (neu-N) transgenic mouse model to study galectin-3 binding on adoptively transferred high avidity neu-specific CD8+ T cells derived from TCR transgenic mice. Galectin-3 binding was found to be associated with increased expression of programmed death 1 (PD1) and apoptosis markers in vivo, and diminished cytokine production in vitro. Abrogation of galectin-3 expression by genetic knock-out in the high avidity CD8 T cells resulted in increased IFN-γ production when compared to wild type controls. Furthermore, we also observed that galectin-3 binding to the T cell surface occurs in an autocrine manner, and is dependant on antigen stimulation as well as sialylation of terminal galactose residues. Using a combination of microarray and co-immunoprecipitation experiments, we have identified several new binding partners through which galectin-3 may mediate its immunosuppressive functions. These studies provide a potential new mechanism by which the function of antigen-specific T cells is down-regulated after trafficking into the tumor micro-environment. Citation Format: Theodore S. Kouo, Melissa Bowman, Lanqing Huang, Todd Armstrong, Elizabeth Jaffee. Regulation of tumor-specific CD8 T cells via interactions between galectin-3 and surface glycoproteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1263. doi:10.1158/1538-7445.AM2013-1263

Abstract 5205: Galectin-3 and AP-1 mediate transcriptional effect of Arylsulfatase B (N-acetylgalactosamine-4-sulfatase) on versican in prostatic cells.

Abstract N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) modifies the glycosaminoglycans (GAGs) chondroitin-4-sulfate (C4S) and dermatan sulfate by removing 4-sulfate groups from the non-reducing end and thereby regulating GAG degradation. Previous studies demonstrated that ARSB was reduced in malignant human colonic, mammary, and prostatic tissue and suggested that ARSB acts as a tumor suppressor. In this report, ARSB was silenced by siRNA in the WPMY-1 human prostate stromal myofibroblast cell line (ATCC), and effects on C4S, total sulfated glycosaminoglycans (GAGs), ErbB2, total EGFR, phospho-ERK, versican, and AP-1 were determined. Total sulfated GAGs were measured by Blyscan assay (Biocolor), and C4S was determined by Blyscan assay following immunoprecipitation by C4S antibody. Phospho(T202/Y204 for ERK1 or T185/Y187 for ERK2)-ERK, ErbB2, and total EGFR were measured by ELISA. Versican, an extracellular matrix chondroitin sulfate proteoglycan with EGF-like repeats at its C-terminus, was measured by ELISA and QPCR. ARSB and galectin-3 were silenced by siRNA (Qiagen). ARSB activity declined from 140.4 ± 8.4 to 21.4 ± 0.9 nmol/mg protein/hr following silencing. Total sulfated GAGs increased from 17.9 ± 0.7 μg/mg protein in control cells to 28.6 ± 1.4 μg/mg protein following ARSB silencing, and C4S increased from 7.4 ± 0.6 μg/mg protein to 15.5 ± 0.8 μg/mg protein following ARSB silencing. Total EGFR increased to 2.65 times baseline, and ErbB2 increased to over six-fold times baseline. Phospho-ERK increased to more than three times baseline, from 5.0 ± 0.5 ng/mg protein to 15.2 ± 0.8 ng/mg protein following silencing. Versican protein increased from 122.1 ± 5.3 ng/mg protein in control to 295.2 ± 11.2 ng/mg protein and mRNA increased more than two-fold. ARSB-silencing induced increases in versican mRNA and in activation of c-Jun and c-Fos, and these increases were inhibited when galectin-3 was also silenced. [All findings are highly significant (p&amp;lt;0.001, one-way ANOVA with Tukey-Kramer post-test)]. Previously, we have shown reduced binding of galectin-3 to C4S following ARSB silencing. Since there is an AP-1 binding site in the versican promoter, study data demonstrate that transcriptional effects of ARSB silencing on versican are mediated by galectin-3 and AP-1. The profound effects of ARSB silencing on versican and phospho-ERK in the prostatic myofibroblasts provide further evidence that ARSB acts as a tumor suppressor. Citation Format: Sumit Bhattacharyya, Leonid Feferman, Joanne Kramer Tobacman. Galectin-3 and AP-1 mediate transcriptional effect of Arylsulfatase B (N-acetylgalactosamine-4-sulfatase) on versican in prostatic cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2013-5205