heparitin-sulfate and Pancreatic-Neoplasms

Necuparanib, a rationally engineered low-molecular-weight heparin, combined with gemcitabine/nab-paclitaxel showed an encouraging safety and oncologic signal in a phase Ib trial. This randomised multicentre phase II trial evaluates the addition of necuparanib or placebo to gemcitabine/nab-paclitaxel in untreated metastatic pancreatic ductal adenocarcinoma (PDAC).. Eligibility included 18 years, histologically or cytologically confirmed metastatic PDAC, measurable disease and Eastern Co-Operative Oncology Group performance status of 0-1. Patients were randomly assigned to necuparanib (5 mg/kg subcutaneous injection once daily) or placebo (subcutaneous injection once daily) and gemcitabine/nab-paclitaxel on days 1, 8 and 15 of 28-day cycles. The primary end-point was median overall survival (OS), and secondary end-points included median progression-free survival, response rates and safety.. One-hundred ten patients were randomised, 62 to necuparanib arm and 58 to placebo arm. The futility boundary was crossed at a planned interim analysis, and the study was terminated by the Data Safety Monitoring Board. The median OS was 10.71 months (95% confidence interval [CI]: 7.95-11.96) for necuparanib arm and 9.99 months (95% CI: 7.85-12.85) for placebo arm (hazard ratio: 1.12, 95% CI: 0.66-1.89, P-value: 0.671). The necuparanib arm had a higher incidence of haematologic toxicity relative to placebo patients (83% and 70%).. The addition of necuparanib to standard of care treatment for advanced PDAC did not improve OS. Safety was acceptable. No further development of necuparanib is planned although targeting the coagulation cascade pathway remains relevant in PDAC. NCT01621243.

Topics: Adult; Aged; Aged, 80 and over; Albumins; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Pancreatic Ductal; Deoxycytidine; Double-Blind Method; Female; Follow-Up Studies; Gemcitabine; Heparitin Sulfate; Humans; Male; Middle Aged; Paclitaxel; Pancreatic Neoplasms; Prognosis; Survival Rate

In pancreatic cancer (PC) progression the protein Annexin A1 (ANXA1) has been described as oncogenic factor. Thus, the need to inhibit its action, mainly the extracellular form, has become an appealing cue for the anti-cancer research. Heparan sulfate (HS) is a glycosaminoglycan of the extracellular matrix known to bind several molecules, as growth factors and cytokines, generating a kind of reservoir in the extracellular environment. Here, we started our study by showing the physical calcium-dependent interaction between HS and ANXA1 as both full-length protein and N-terminal portion, Ac2-26 by biophysical techniques. HS is able to inhibit the migration/invasion process of human PC MIA PaCa-2 cells and partially revert their mesenchymal phenotype as reported through the expression of specific protein markers and the growth in colonies and in 3D-spheroids. Furthermore, both on MIA PaCa-2 and PANC-1 cells, HS blocks the effects of Ac2-26, which enhances the aggressive behavior of PC cells if added alone. These effects appear evident also on endothelial cells whose activation is promoted by Ac2-26 but not in presence of HS. Thus, the interference of the interaction ANXA1-HS on angiogenesis strongly emerges. Moreover, once sequestered by HS, ANXA1 is not more able to bind the formil-peptide receptors (FPRs) preventing the increase of calcium mobilization, peculiar for cell motility. These findings introduce a new important tale in the knowledge about the inhibition of the ANXA1 action in PC development. Further information will be useful to highlight the interaction of HS with the protein, focusing on the characterization of the glycosaminoglycan and on in vivo assays.

Topics: Annexin A1; Cell Line, Transformed; Cell Line, Tumor; Cell Movement; Extracellular Fluid; Heparitin Sulfate; Human Umbilical Vein Endothelial Cells; Humans; Pancreatic Neoplasms; Protein Binding

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.Heparan sulfate (HS) of specific length, i.e., hexasaccharide (HS06), but not longer or shorter sequences, selectively inhibit cancer stem cells (CSCs) through isoform specific activation of p38 mitogen-activated protein kinase. These findings will have major implication for developing chemical probes to decipher complex signaling events that govern cancer stem cells. Additionally, there are direct implications for designing glycosaminoglycan based cancer therapies to selectively target CSCs that escape killing by traditional chemotherapy threatening cancer relapse.

Topics: Breast Neoplasms; Cell Self Renewal; Colorectal Neoplasms; Drug Discovery; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Heparitin Sulfate; Humans; Neoplastic Stem Cells; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Signal Transduction; Transcription Factor 4; Tumor Cells, Cultured

All Hedgehog morphogens are released from producing cells, despite being synthesized as N- and C-terminally lipidated molecules, a modification that firmly tethers them to the cell membrane. We have previously shown that proteolytic removal of both lipidated peptides, called shedding, releases bioactive Sonic hedgehog (Shh) morphogens from the surface of transfected Bosc23 cells. Using in vivo knockdown together with in vitro cell culture studies, we now show that glypican heparan sulfate proteoglycans regulate this process, through their heparan sulfate chains, in a cell autonomous manner. Heparan sulfate specifically modifies Shh processing at the cell surface, and purified glycosaminoglycans enhance the proteolytic removal of N- and C-terminal Shh peptides under cell-free conditions. The most likely explanation for these observations is direct Shh processing in the extracellular compartment, suggesting that heparan sulfate acts as a scaffold or activator for Shh ligands and the factors required for their turnover. We also show that purified heparan sulfate isolated from specific cell types and tissues mediates the release of bioactive Shh from pancreatic cancer cells, revealing a previously unknown regulatory role for these versatile molecules in a pathological context.

Topics: Animals; Blotting, Western; Body Patterning; Cell Membrane; Cells, Cultured; Drosophila melanogaster; Drosophila Proteins; Glypicans; Hedgehog Proteins; Heparitin Sulfate; Humans; Mice; Pancreatic Neoplasms; Protein Processing, Post-Translational; Proteolysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Epithelial-mesenchymal transition (EMT) is a critical early event in tumorigenesis. The contribution of heparan sulfate (HS) to EMT has not been fully elucidated. HS D-glucosaminyl 3-O-sulfotransferase-3B1 (3-OST-3B1) participates in the final step of HS fine structure biosynthesis, whose involvement in cancer has yet to be determined. This study demonstrated that following treatment with trichostatin-A, a histone deacetylase inhibitor, 3-OST-3B1 gene expression was activated in the pancreatic cancer cell line, PANC-1. By chromatin immunoprecipitation analysis, permissive histone modifications including an increase in histone H3 lysine 9 monoactylation (H3 ac K9) but a decrease in methylated histone H3 (H3 me K9) were observed accompanying transcriptional activation of 3-OST-3B1. Functional, results revealed that increased 3-OST-3B1 levels were involved in the promotion of EMT processes. In vitro studies demonstrated that overexpression of 3-OST-3B1 in both pancreatic cancer cells and vascular endothelial cells could trigger an EMT-like phenotype as evidenced by the up-regulation of Snail at the mRNA and protein level, and its nuclear translocation. And 3-OST-3B1 appeared to be sufficient for the development of a more mesenchymal phenotype in vivo. Together, the results from this study unveiled a distinct function for 3-OST-3B1 as an EMT inducer in cancer and provided a link between histone modification and EMT modulation.

Topics: Azacitidine; Base Sequence; Cell Line, Tumor; Chromatin Immunoprecipitation; Decitabine; Endothelial Cells; Epithelial-Mesenchymal Transition; Heparitin Sulfate; Histones; Humans; Hydroxamic Acids; Methylation; Pancreatic Neoplasms; RNA, Messenger; Sequence Analysis, DNA; Snail Family Transcription Factors; Sulfotransferases; Transcription Factors; Transcriptional Activation

Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparin-binding domain for signaling was assessed. It was determined that key residues in human (h) Shh involved in heparin and HSPG syndecan-4 binding and biological activity included the well known cationic Cardin-Weintraub motif (lysines 32-38) but also a previously unidentified major role for lysine 178. The activity of Shh mutated in these residues was tested by quantitation of alkaline phosphatase activity in C3H10T1/2 cells differentiating into osteoblasts and hShh-inducible gene expression in PANC1 human pancreatic ductal adenocarcinoma cells. Mutated hShhs such as K37S/K38S, K178S, and particularly K37S/K38S/K178S that could not interact with heparin efficiently had reduced signaling activity compared with wild type hShh or a control mutation (K74S). In addition, the mutant hShh proteins supported reduced proliferation and invasion of PANC1 cells compared with control hShh proteins, following endogenous hShh depletion by RNAi knockdown. The data correlated with reduced Shh multimerization where the Lys-37/38 and/or Lys-178 mutations were examined. These studies provide a new insight into the functional roles of hShh interactions with HSPGs, which may allow targeting this aspect of hShh biology in, for example, pancreatic ductal adenocarcinoma.

Topics: Adenocarcinoma; Amino Acid Motifs; Amino Acid Substitution; Cell Line, Tumor; Hedgehog Proteins; Heparitin Sulfate; Humans; Mutation, Missense; Osteoblasts; Pancreatic Neoplasms; Protein Multimerization; Protein Structure, Tertiary; Signal Transduction; Syndecan-4

NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding Sites; Carcinoma, Ductal; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Epitopes; HeLa Cells; Heparitin Sulfate; Humans; Immunoglobulins; Interferon-gamma; Killer Cells, Natural; Ligands; Male; Melanoma; Molecular Sequence Data; Mutation; Natural Cytotoxicity Triggering Receptor 2; Pancreatic Neoplasms; Prostatic Neoplasms; Protein Binding; Protein Structure, Tertiary; Receptors, Immunologic; Recombinant Fusion Proteins

Heparan sulfate proteoglycans are integral components of the extracellular matrix that surrounds all mammalian cells. In addition to providing structural integrity, they act as a storage depot for a variety of heparan sulfate (HS)-binding proteins, including growth factors and chemokines. Heparanase is a matrix-degrading enzyme that cleaves heparan sulfate side chains from the core proteoglycans, thus liberating such HS-binding proteins, as well as potentially contributing to extracellular matrix degradation. Here, we report that heparanase mRNA and protein expression are increased in the neoplastic stages progressively unfolding in a mouse model of multistage pancreatic islet carcinogenesis. Notably, heparanase is delivered to the neoplastic lesions in large part by infiltrating Gr1+/Mac1+ innate immune cells. A sulfated oligosaccharide mimetic of heparan sulfate, PI-88, was used to inhibit simultaneously both heparanase activity and HS effector functions. PI-88 had significant effects at distinct stages of tumorigenesis, producing a reduction in the number of early progenitor lesions and an impairment of tumor growth at later stages. These responses were associated with decreased cell proliferation, increased apoptosis, impaired angiogenesis, and a substantive reduction in the number of invasive carcinomas. In addition, we show that the reduction in tumor angiogenesis is correlated with a reduced association of VEGF-A with its receptor VEGF-R2 on the tumor endothelium, implicating heparanase in the mobilization of matrix-associated VEGF. These data encourage clinical applications of inhibitors such as PI-88 for the many human cancers where heparanase expression is elevated or mobilization of HS-binding regulatory factors is implicated.

Topics: Amino Acid Sequence; Animals; Capillaries; Cattle; Disease Models, Animal; Endothelium, Vascular; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glucuronidase; Heparitin Sulfate; Islets of Langerhans; Mice; Molecular Sequence Data; Neovascularization, Pathologic; Pancreatic Neoplasms; Peptide Fragments; Polymerase Chain Reaction

The amount and the types of glycosaminoglycans (GAGs) present in human pancreatic carcinoma were examined and compared with those in normal pancreas. Human pancreatic carcinoma contained increased levels (4-fold) of total GAGs. Particularly, this carcinoma is characterized by a 12-fold increase of hyaluronan (HA) and a 22-fold increase in chondroitin sulfate (CS) content. CS in pancreatic carcinoma exhibited an altered disaccharide composition which is associated with marked increase of non-sulfated and 6-sulfated disaccharides. Dermatan sulfate (DS) was also increased (1.5-fold) in carcinoma, whereas heparan sulfate (HS), the major GAG of normal pancreas, becomes the minor GAG in pancreatic carcinoma without significant changes in the content and in molecular size. In all cases, the galactosaminoglycans (GalGAGs, i.e. CS and DS) derived from pancreatic carcinomas were of lower molecular size compared to those from normal pancreas. The results in this study indicate, for the first time, that human pancreatic carcinoma is characterized by highly increased amounts of HA and of a structurally altered CS.

Topics: Carbohydrate Sequence; Chondroitin Sulfates; Dermatan Sulfate; Disaccharides; Heparitin Sulfate; Humans; Hyaluronic Acid; Molecular Weight; Pancreas; Pancreatic Neoplasms; Polysaccharides

Structural features of heparin potentially important for heparanase-inhibitory activity were examined by measuring the ability of heparin derivatives to affect the degradation of [3H]acetylated heparan sulphate by tumor cell heparanases. IC50 values were determined using an assay which distinguished degraded from undegraded substrate by precipitation of the latter with cetylpyridinium chloride (CPC). Removal of heparin's 2-O-sulphate and 3-O-sulphate groups enhanced heparanase-inhibitory activity (50%). Removal of its carboxyl groups slightly lowered the activity (18%), while combining the treatments abolished the activity. At least one negative charge on the iduronic acid/idose moiety, therefore, is necessary for heparanase-inhibitory activity. Replacing heparin's N-sulphate groups with N-acetyl groups reduced its activity (37%). Comparing this heparin derivative with 2,3-O-desulphated heparin, the placement of sulphate groups appears important for activity since the two structures have similar nominal linear charge density. In addition, unsubstituted uronic acids are nonessential for inhibition since their modification (periodate-oxidation/borohydride-reduction) enhanced rather than reduced heparanase-inhibitory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohydride-reduced] heparin) were tested in the chick chorioallantoic membrane (CAM) bioassay for anti-angiogenic activity and found to be at least as efficacious as heparin. 2,3-O-desulphated heparin also significantly decreased the tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay.

Topics: Animals; Anticoagulants; Antineoplastic Agents; Chick Embryo; Chondroitin Sulfates; Enzyme Inhibitors; Female; Glucuronidase; Glycoside Hydrolases; Heparin; Heparitin Sulfate; Humans; Lung Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Nude; Molecular Structure; Neoplasm Metastasis; Neoplasms, Experimental; Neovascularization, Physiologic; Pancreatic Neoplasms

The prognosis for carcinoma of the pancreas is extremely poor. One of the characteristics of this tumor is its invasion of the surrounding tissues. Reduction of glycoprotein is considered to be conducive to invasion of the basement membrane by carcinoma cells. Heparan sulfate proteoglycan (HSPG), a kind of glycoprotein, is an important component of basement membrane. In this study, the relation between HSPG and carcinoma of the pancreas was examined by using the immunohistochemical method, and the survival rate of pancreatic adenocarcinoma was evaluated. We found that some carcinomas contained little or no HSPG. The poorer the differentiation of an adenocarcinoma of the pancreas, the lower was its content of HSPG. The level of HSPG was significantly different in carcinomatous and in noncarcinomatous cells. There was a close correlation among the content of HSPG, the degree of differentiation of carcinomas of the pancreas, and the survival time. HSPG seems to be useful in prognosis of adenocarcinoma of the pancreas.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Female; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunohistochemistry; Male; Middle Aged; Pancreatic Neoplasms; Prognosis; Proteoglycans; Survival Rate