Mark M. Fuster

The sweet and sour of cancer: glycans as novel therapeutic targets.

A growing body of evidence supports crucial roles for glycans at various pathophysiological steps of tumour progression. Glycans regulate tumour proliferation, invasion, haematogenous metastasis and angiogenesis, and increased understanding of these roles sets the stage for developing pharmaceutical agents that target these molecules. Such novel agents might be used alone or in combination with operative and/or chemoradiation strategies for treating cancer.

Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses.

Here we have studied the involvement of endothelial heparan sulfate in inflammation by inactivating the enzyme N-acetyl glucosamine N-deacetylase–N-sulfotransferase-1 in endothelial cells and leukocytes, which is required for the addition of sulfate to the heparin sulfate chains. Mutant mice developed normally but showed impaired neutrophil infiltration in various inflammation models. These effects were due to changes in heparan sulfate specifically in endothelial cells. Decreased neutrophil infiltration was partially due to altered rolling velocity correlated with weaker binding of L-selectin to endothelial cells. Chemokine transcytosis across endothelial cells and presentation on the cell surface were also reduced, resulting in decreased neutrophil firm adhesion and migration. Thus, endothelial heparan sulfate has three functions in inflammation: by acting as a ligand for L-selectin during neutrophil rolling; in chemokine transcytosis; and by binding and presenting chemokines at the lumenal surface of the endothelium.

Broad targeting of angiogenesis for cancer prevention and therapy

Deregulation of angiogenesis – the growth of new blood vessels from an existing vasculature – is a main driving force in many severe human diseases including cancer. As such, tumor angiogenesis is important for delivering oxygen and nutrients to growing tumors, and therefore considered an essential pathologic feature of cancer, while also playing a key role in enabling other aspects of tumor pathology such as metabolic deregulation and tumor dissemination/metastasis. Recently, inhibition of tumor angiogenesis has become a clinical anti-cancer strategy in line with chemotherapy, radiotherapy and surgery, which underscore the critical importance of the angiogenic switch during early tumor development. Unfortunately the clinically approved anti-angiogenic drugs in use today are only effective in a subset of the patients, and many who initially respond develop resistance over time. Also, some of the anti-angiogenic drugs are toxic and it would be of great importance to identify alternative compounds, which could overcome these drawbacks and limitations of the currently available therapy. Finding “the most important target” may, however, prove a very challenging approach as the tumor environment is highly diverse, consisting of many different cell types, all of which may contribute to tumor angiogenesis. Furthermore, the tumor cells themselves are genetically unstable, leading to a progressive increase in the number of different angiogenic factors produced as the cancer progresses to advanced stages. As an alternative approach to targeted therapy, options to broadly interfere with angiogenic signals by a mixture of non-toxic natural compound with pleiotropic actions were viewed by this team as an opportunity to develop a complementary anti-angiogenesis treatment option. As a part of the “Halifax Project” within the “Getting to know cancer” framework, we have here, based on a thorough review of the literature, identified 10 important aspects of tumor angiogenesis and the pathological tumor vasculature which would be well suited as targets for anti-angiogenic therapy: (1) endothelial cell migration/tip cell formation, (2) structural abnormalities of tumor vessels, (3) hypoxia, (4) lymphangiogenesis, (5) elevated interstitial fluid pressure, (6) poor perfusion, (7) disrupted circadian rhythms, (8) tumor promoting inflammation, (9) tumor promoting fibroblasts and (10) tumor cell metabolism/acidosis. Following this analysis, we scrutinized the available literature on broadly acting anti-angiogenic natural products, with a focus on finding qualitative information on phytochemicals which could inhibit these targets and came up with 10 prototypical phytochemical compounds: (1) oleanolic acid, (2) tripterine, (3) silibinin, (4) curcumin, (5) epigallocatechin-gallate, (6) kaempferol, (7) melatonin, (8) enterolactone, (9) withaferin A and (10) resveratrol. We suggest that these plant-derived compounds could be combined to constitute a broader acting and more effective inhibitory cocktail at doses that would not be likely to cause excessive toxicity. All the targets and phytochemical approaches were further cross-validated against their effects on other essential tumorigenic pathways (based on the “hallmarks” of cancer) in order to discover possible synergies or potentially harmful interactions, and were found to generally also have positive involvement in/effects on these other aspects of tumor biology. The aim is that this discussion could lead to the selection of combinations of such anti-angiogenic compounds which could be used in potent anti-tumor cocktails, for enhanced therapeutic efficacy, reduced toxicity and circumvention of single-agent anti-angiogenic resistance, as well as for possible use in primary or secondary cancer prevention strategies.

Tumor attenuation by combined heparan sulfate and polyamine depletion

Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemble de novo through a pathway sensitive to the inhibitor, α-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferation in vitro and in vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.

Surfen, a small molecule antagonist of heparan sulfate

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF165-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.

Heparan sulfate regulates VEGF165 And VEGF121-mediated vascular hyperpermeability

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF165 and VEGF121. Because VEGF121 does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF165 and VEGF121. Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF165 and VEGF121, suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.

Expression Patterns of α2,3-Sialyltransferases and α1,3-Fucosyltransferases Determine the Mode of Sialyl Lewis X Inhibition by Disaccharide Decoys

A variety of human adenocarcinomas express sialylated, fucosylated Lewis blood group antigens on cell surface and secreted mucins. Binding of these antigens to P-selectin on platelets is thought to facilitate formation of platelet-tumor emboli in the circulation, which in turn allows sequestration of the tumor cells in the microvasculature. Here we report a pharmacologic approach for blocking these interactions through metabolic inhibition of sialylation. Peracetylated forms of Galβ1,4GlcNAcβ-O-naphthalenemethanol and GlcNAcβ1,3Galβ-O-naphthalenemethanol were taken up by LS180 human colon carcinoma cells, O-deacetylated, and utilized as biosynthetic intermediates, resulting in heterogeneous oligosaccharides. The primed oligosaccharides included sialylated, sulfated, and fucosylated products based on mass spectrometry. Assembly of free oligosaccharides on the glycosides decoyed glycosylation of cellular glycoproteins, as assessed by altered binding of lectins and carbohydrate-specific antibodies. Expression of α2,3-sialylated oligosaccharides on the cell surface was diminished specifically, whereas α2,6-sialylation and fucosylation were not. In U937 lymphoma cells, the glycosides decreased fucosylation without affecting sialylation. The differential inhibitory activities correlated inversely with fucosyltransferase and sialyltransferase activity based on enzyme assays and microarray analysis. Regardless of the mechanism, the disaccharides blocked the cells from forming selectin ligands and inhibited adhesion to immobilized selectins, suggesting that the glycosides might prove useful for interfering with tumor cell adhesion and metastasis.

Locoregional and distant failure following image-guided stereotactic body radiation for early-stage primary lung cancer

PURPOSE To report our institutional experience using image-guided stereotactic body radiation therapy (SBRT) for early stage lung cancer, including an analysis into factors associated with nodal and distant failures (NF, DF). METHODS Forty-eight patients with early-stage primary lung cancer were treated with image-guided SBRT between 2007 and 2009. Median prescription dose was 48 Gy in 4 fractions. Toxicity was graded according to the NCI CTCAE v3.0 scale. RESULTS Local failure was detected in two lesions and actuarial 24-month local control was 95%. At 24 months, the cumulative incidence of NF was 6%, and DF was 29%. Larger lesions (>3 cm) and younger age (<70 years) were the only factors found to be significantly correlated with increased DF (p=0.005 and p=0.015, respectively). A single grade ≥ 3 toxicity was observed. After adjusting for age and lesion size, distant failure was significantly associated with a poorer OS (Cox regression, p=0.0059). CONCLUSION Image-guided SBRT can produce excellent LC rates with minimal toxicity. Distant failure was a major determinant of OS and the most common pattern of failure, indicating a potential role for systemic therapy in younger patients with large lesions.

Endothelial Heparan Sulfate in Angiogenesis

Heparan sulfate (HS) is a linear polysaccharide composed of 50-200 glucosamine and uronic acid (glucuronic acid or iduronic acid) disaccharide repeats with epimerization and various sulfation modifications. HS is covalently attached to core proteins to form HS-proteoglycans. Most of the functions of HS-proteoglycans are mediated by their HS moieties. The biosynthesis of HS is initiated by chain polymerization and is followed by stepwise modification reactions, including sulfation and epimerization. These modifications generate ligand-binding sites that modulate cell functions and activities of proteinases and/or proteinase inhibitors. HS is abundantly expressed in developing and mature vasculature, and understanding its roles in vascular biology and related human diseases is an area of intense investigation. In this chapter, we summarize the significant recent advances in our understanding of the roles of HS in developmental and pathological angiogenesis with a major focus on studies using transgenic as well as gene knockout/knockdown models in mice and zebrafish. These studies have revealed that HS critically regulates angiogenesis by playing a proangiogenic role, and this regulatory function critically depends on HS fine structure. The latter is responsible for facilitating cell-surface binding of various proangiogenic growth factors that in turn mediate endothelial growth signaling. In cancer, mouse studies have revealed important roles for endothelial cell-surface HS as well as matrix-associated HS, wherein signaling by multiple growth factors as well as matrix storage of growth factors may be regulated by HS. We also discuss important mediators that may fine-tune such regulation, such as heparanase and sulfatases; and models wherein targeting HS (or core protein) biosynthesis may affect tumor growth and vascularization. Finally, the importance of targeting HS in other human diseases wherein angiogenesis may play pathophysiologic (or even therapeutic) roles is considered.

Deficiency of Endothelial Heparan Sulfates Attenuates Allergic Airway Inflammation

The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1f/fTekCre+ (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-β1, and eotaxin levels, as well as decreased expression of TGF-β1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1f/fTekCre− wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-β1.