Presentation on theme: "Essentials of Glycobiology Ajit Varki Lecture 35"— Presentation transcript:
1 Essentials of Glycobiology Ajit Varki Lecture 35 Changes in Glycosylation in Cancer
2 CLINICALLY RELEVANT CANCER INVOLVES A MULTISTEP PROCESS INHERITED GENOMIC DNA ABNORMALITIESDNA DAMAGE CAUSED BY EXTERNAL AGENTSIMMORTALIZATION OF GROWTH POTENTIALABNORMAL ONCOGENE EXPRESSION CAUSING INCREASED GROWTHLOSS OF TUMOR SUPPRESSOR GENES THAT NORMALLY CONTROL SUCH CELLSACCUMULATION OF GENETIC ABNORMALITIESDEVELOPMENT OF GENETIC HETEROGENEITY, ALLLOWING TUMOR SPREAD (METASTASIS)METASTASIS IS NOW THE MAIN CAUSE OF DEATH IN HUMANS WITH CANCER
3 Clonal Expansion and Growth Angiogenesis Invasion of Basement Membrane Passage through Extracellullar MatrixIntravasationTumor Cell interactionswith Blood cellsAdhesion to endotheliumInvasion of basement membraneExtravasationGrowth and Angiogenesis
4 Historical Background 1950s: Enhanced binding of certain plant lectins (e.g., wheat germ agglutinin) to animal tumor cells1960s: In vitro transformation of cells frequently accompanied by increases in overall size of metabolically labelled glycopeptides.s: Search for “magic bullet” monoclonal antibodies against cancer. Many “tumor-specific” antibodies directed against carbohydrate epitopes, especially on glycosphingolipids.s: These epitopes are actually “oncofetal antigens” - also expressed in embryonic tissues, and in a few normal adult cell types
5 Historical Background (Continued) s: Significant correlations between certain types of altered glycosylation and actual prognosis of tumor-bearing animals or patients.1990s: Gene transfection experiments show that glycosylation changes may indeed be critical to aspects of tumor cell behavior.Late 1990s-2004: proof using mice with genetically altered glycosylation or glycan binding proteins.Late 1990s-2004: Diagnostic applications in humans, therapeutic applications in mice.Therapeutic applications in Humans are still few!
6 Altered glycosylation is an universal feature of tumor cells. Changes typically seen are highly selective and specific.Cancer Cells are genetically heterogenous and are constantly undergoing Micro-evolution (“Survival of the Fittest”).Thus, there is likely selection for highly specific changes seen in natural tumors.
7 General Ways In Which Glycosylation Can Be Altered in Malignant Cells Loss of expression of certain structuresExcessive expression of certain structuresPersistence of incomplete or truncated structuresAccumulation of precursorsAppearance of new structures.Note: Changes in early branch points in pathways can markedly decrease amount of one class of structures, while causing dominance of another.Only a limited subset of biosynthetic pathways are frequently correlated with malignant transformation and tumor progression
8 Changes in Core Structures and Core Proteins N-glycans: Altered Branching, especially over-expression of GlcNAc Transferase V (GNT-V)O-glycans: Dominance of truncated structures (T, Tn, sialyl-Tn). Excessive production and shedding of mucinsGlycosphingolipids: Over-expression and shedding, especially in neuroectodermal tumors (melanoma, neuroblastoma etc.)Glycosaminoglycans: Altered expression, especially Hyaluronan (ligand for CD44). Altered expression of some Heparan Sulfate ProteoglycansGPI Anchors - losses in some leukemias.
9 Changes in Shared Outer Structures Increased outer chain alpha1-3(4) Fucosylation, generating Selectin ligandsIncreased Expression of Polylactosamines (Galectin Ligands)Altered Expression of ABO Blood Group StructuresIncrease in overall Sialic Acid contentEnhanced expression of some Sialic acid linkagesAlterations in Types of Sialic Acids
10 Major Glycan Classes in Animal Cells HYALURONANGLYCOSAMINO-GLYCANSHEPARAN SULFATECHONDROITINSULFATEMajor Glycan Classes in Animal CellsPSSSSer-O-SSSSS-O-SerNSNSProteoglycanAcN-LINKED CHAINSO-LINKEDCHAINGLYCOPHOSPHO-LIPIDANCHORPSEtnPONNSer/ThrAsnAsnNH2GLYCOSPHINGOLIPIDGlycoproteinINOSITOLAcOUTSIDEPSialic AcidsINSIDEO-LINKED GlcNAcOSer
11 Altered N-Linked Glycosylation in Tumor cells due to Overexpression of GNT-V
12 Altered N-Linked Glycosylation in Tumor cells due to Overexpression of GNT-V
13 Increased GlcNAc Transferase-V mediated 1-6 Branching of N-glycans Transcriptionally increased expression of GNT-V induced by viral and chemical carcinogenesisCell lines with increased GNT-V expression show increased frequency of metastasisSpontaneous revertants for enzyme expression lose metastatic phenotypeClinical specimens of some tumors show increased staining with the lectin L-PHA, which preferentially recognizes 1-6 branched N-glycans
14 Increased GlcNAc Transferase-V mediated 1-6 Branching of N-glycans Transfection of GNT-V cDNA into cells causes:Visually obvious transformed phenotypeEnhanced colony formation in agarIncreased cell spreadingEnhanced invasiveness through membranesTumorigenic behavior in previously non-tumorigenic cells.By conventional criteria, these are characteristics of a true oncogene!
15 Suppression of Tumor growth and metastasis in (Mgat5) GlcNAcT-V-deficient mice Mgat5 (-/-) mice lack GlcNAcT-V products and appear fertile/normalAltered proliferation in lymphocytes and epitheliumMammary tumor growth and metastases induced by the polyomavirus middle T oncogene considerably decreasedGNT-V stimulates membrane ruffling and Pl-3 kinase-protein kinase B activation - a positive feedback loop that amplifies oncogene signaling and tumor growth in vivo.Inhibitors of GNT-V could be useful in treatment by targeting dependency on focal adhesion and signaling for growth and metastasis
16 Increased 1-6 Branching of N-glycans in Cancer Precise mechanism(s) of biological outcomes unknownIncreased polyllactosamines recognized by galectins?Increased outer chain polyfucosylation and sialyl Lewis X production -recognized by the selectins?General physical effect of branching itself? 1-6 branch has a "broken wing" conformation, perhaps directly associating the glycan with nearby peptide moeity?Altered functioning of glycosylated adhesion molecules like integrins and/or cadherins?Metabolic inhibition of N-glycan processing by swainsonine has the converse effects. Swainsonine now in clinical trials for patients with advanced cancer.
17 Major Glycan Classes in Animal Cells HYALURONANGLYCOSAMINO-GLYCANSHEPARAN SULFATECHONDROITINSULFATEMajor Glycan Classes in Animal CellsPSSSSer-O-SSSSS-O-SerNSNSProteoglycanAcN-LINKED CHAINSO-LINKEDCHAINGLYCOPHOSPHO-LIPIDANCHORPSEtnPONNSer/ThrAsnAsnNH2GLYCOSPHINGOLIPIDGlycoproteinINOSITOLAcOUTSIDEPSialic AcidsINSIDEO-LINKED GlcNAcOSer
18 Altered Expression/Shedding of Glycosphingolipids Many "tumor-specific" monoclonal antibodies raised against tumors recognize glycosphingolipidsSome highly enriched in specific types of tumors e.g., Gb3/CD77 in Burkitt’s lymphoma and GD3 in melanomasSome tumors (particularly melanoma and neuroblastoma) synthesize very high levels of gangliosidesSome, e.g., GD2 and GM2 not found at high levels in extraneural cells - targets for immunotherapyIn vitro studies suggest that some gangliosides can affect growth controlLarge quantities of gangliosides "shed" by some tumors have strong immunosuppressive effects
19 Major Glycan Classes in Animal Cells HYALURONANGLYCOSAMINO-GLYCANSHEPARAN SULFATECHONDROITINSULFATEMajor Glycan Classes in Animal CellsPSSSSer-O-SSSSS-O-SerNSNSProteoglycanAcN-LINKED CHAINSO-LINKEDCHAINGLYCOPHOSPHO-LIPIDANCHORPSEtnPONNSer/ThrAsnAsnNH2GLYCOSPHINGOLIPIDGlycoproteinINOSITOLAcOUTSIDEPSialic AcidsINSIDEO-LINKED GlcNAcOSer
20 MUCINS IN NORMAL AND MALIGNANT EPITHELIUM Altered sugar chainsCANCERMucinsNORMALBASEMENTMEMBRANE
21 Mucins with Altered O-Glycosylation in Cancer Mucins are major carriers of altered glycosylationMucin expression correlates with metastatic potential in some carcinomasShed Mucins in body fluids have diagnostic valueMucins can block adhesion by cytolytic cells, e.g., NK cells.Mucins can show incomplete O-glycosylation
22 Truncated O-glycans on Mucins in Cancer Correlation between T and Tn expression, spontaneous antibodies directed against them, and prognosisThe most extreme form of underglycosylation results in expression of "naked" mucin polypeptidesClinical trials underway to provoke/enhance these immune responses by injecting patients with peptide antigens, sometimes bearing multiple copies Tn of Sialyl-TnEarly results promising
23 Major Glycan Classes in Animal Cells HYALURONANGLYCOSAMINO-GLYCANSHEPARAN SULFATECHONDROITINSULFATEMajor Glycan Classes in Animal CellsPSSSSer-O-SSSSS-O-SerNSNSProteoglycanAcN-LINKED CHAINSO-LINKEDCHAINGLYCOPHOSPHO-LIPIDANCHORPSEtnPONNSer/ThrAsnAsnNH2GLYCOSPHINGOLIPIDGlycoproteinINOSITOLAcOUTSIDEPSialic AcidsINSIDEO-LINKED GlcNAcOSer
24 Hyaluronan (HA) in Cancer Epithelial tumors surrounded by stroma enriched in HA.Corresponding normal epithelia give very low signal for HAExtent of stromal HA accumulation -a strong, independent, negative predictor of survival in many cancersThree major molecular characteristics of HA contribute to normal and tumor cell behaviorUnique hydrodynamic propertiesInteractions with HA-binding “hyaladherins” in the assembly of pericellular and extracellular matrices.Effects on cell signaling and behavior.
25 Hyaluronan in CancerExtracellular matrix surrounding proliferating and migrating cells in embryonic development, regeneration, healing, cancer and vascular disease is highly enriched in HASimple interpretation: HA creates fluid, malleable matrix in which cells can change shape or migrate.HA synthase activity peaks at mitosis. Inhibition of HA synthesis causes cell cycle arrest, just before cell roundingHA interaction with hyaladherins (versican, aggrecan, TSG-6 etc.) in matrix creates microenvironment that supports and promotes dividing and migrating cells.HA interacts with cells by:binding to surface receptors, (e.g., CD44,RHAMM) giving signal transduction and cytoskeletal rearrangementsbeing deposited in cytoplasm? Several intracellular hyaladherins, e.g. Cdc37, IHABP4, an intracellular form of RHAMM are known
26 Hyaluronan in Cancer - evidence in animal models HA overexpression promotes growth of fibrosarcoma and prostate carcinoma and mammary carcinoma metastasis.Overexpression of soluble soluble CD44, RHAMM, or other hyaladherins displaces endogenous HA from its receptors, and inhibits tumor growth and metastasis.Soluble hyaladherins cause loss of HA-induced clustering of plasma membrane CD44, which normally docks gelatinase B (MMP-9) on surface of malignant cells - which promotes tumor cell invasiveness and angiogenesisSoluble CD44 induces G1 arrest or apoptosis in tumor cells and inhibition of MMP-mediated invasion.
27 Hyaluronan in Cancer - evidence in animal models HA oligosaccharides inhibit in vivo tumor growth, presumably by competing for endogenous interactions. HA oligomers also induce G1 arrest or apoptosis in tumor cells - inhibition of the PI 3-kinase-Akt survival pathway?Some tumor cells have increased hyaluronidase and ability to internalize and degrade HA. Penetration of HA-rich stroma or production of angiogenic HA breakdown products may promote progression.HA-RHAMM interactions involved in Ras and signal-regulated kinase signaling pathways. Suppression inhibits cell locomotion and proliferation in vitro and inhibition of tumor growth in vivoOverexpression of RHAMM leads to enhanced tumor growth and metastasis
28 Major Glycan Classes in Animal Cells HYALURONANGLYCOSAMINO-GLYCANSHEPARAN SULFATECHONDROITINSULFATEMajor Glycan Classes in Animal CellsPSSSSer-O-SSSSS-O-SerNSNSProteoglycanAcN-LINKED CHAINSO-LINKEDCHAINGLYCOPHOSPHO-LIPIDANCHORPSEtnPONNSer/ThrAsnAsnNH2GLYCOSPHINGOLIPIDGlycoproteinINOSITOLAcOUTSIDEPSialic AcidsINSIDEO-LINKED GlcNAcOSer
29 Sulfated Glycosaminoglycans in Cancer Tumor metastasis hardly ever occurs in cartilage, which is very rich in Chondroitin Sulfate - mechanisms unknown (apparent basis for the huge market in shark cartilage!)Matrix HS-GAGs act a barrier to invasion and metastasis,HS-GAGS on tumors could play a facilitatory role, by recruiting and/or stabilize growth factors or matrix metalloproteases, or promoting angiogenesis.CHO mutants with decreased HS-GAG production do not form tumors in nude mice. Mechanism may involve change in humoral immune response and/or change of a “salvage” polyamine transport system
30 Multiple Hereditary Exostosis Autosomal dominant traitBony outgrowths (exostoses) usually located next to epiphyseal growth plateSkeletal deformities such as shortened or curved limbs, reduced staturePain caused by muscle and nerve compression% predilection for progression to malignant chondrosarcoma, i.e., a “tumor-suppressor gene”
31 Multiple Hereditary Exostosis Patients with Multiple Hereditary Exostosis have mutations in EXT1 and EXT2The heparan sulfate copolymerase consists of an heterooligomer of EXT1 and EXT2GlcA and GlcNAc transferase active sites of the copolymerase reside in separate subdomainsSeveral mutations in EXT1 and EXT2 can result in deficient heparan sulfate synthesisHeterozygous state produce hereditary exostosisNote: homozygous null state is lethal and mice, and not seen in living humans
32 Common Outer Chains Shared by Different Classes of Glycans = Sialic acidSN-LINKED CHAINONSer/ThrAsnO-LINKED CHAINSecreted ProteinGLYCOSPHINGOLIPIDSONSer/ThrMembrane ProteinAsnOUTSIDECELLMEMBRANEINSIDE
33 Prognostic significance of Sialyl Lewisx expression in patients with colon adenocarcinoma from Nakamori et.al, 1997Siaa2-3Galb1-4GlcNAcb1-31aFucSLex expression also associated with poor prognosis in carcinomas of Breast, Lung, Pancreas, Bladder, Prostate, Biliary tree and OvaryDO SELECTINS PLAY IN ROLE IN ADENOCARCINOMA PROGRESSION?
34 Interactions between Carcinoma Mucins and Selectins Tissue Factor?Resting plateletThrombinAPtLeukocyteActivatedCarcinomaL-selectinP-selectinplateletcellLPAPtLeukocyteSolubleMucinMembrane-bound MucinEE-selectinActivated EndotheliumSelectin Binding sites
35 Effects of Selectin Deficiencies on the Metastatic Progression of GFP-expressing MC-38 Mouse Adenocarcinoma in Syngeneic MiceP- and L-selectin deficiency are additiveP-selectin deficiency works by preventing tumor cell interactions with blood plateletsMechanism(s) of metastasis reduction by L-selectin deficiency unknownE-selectin may also play a role
36 Perioperative Heparin Therapy Reduces Late Deaths from Metastatic Cancer ControlHeparin (Others)Control (Others)Heparin (Cancer)Control (Cancer)Kakkar et al. Int.J.Oncol. 6:885, A retrospective analysis of 1250 patients randomized for peri-operative heparin prophylaxis against venous thrombosis
37 Diverse Effects of Heparin in Cancer Blockade of P- and L-selectinInhibition of the Clotting CascadeInteractions with IntegrinsBinding up of Growth factorsInhibition of AngiogenesisInhibition of HeparanasesAlterations of Protease actionsLots of clinical heparin (standardized for anticoagulation) may vary in the other actionsVery few studies show a detrimental consequence of heparin in murine or human cancer
38 Galectins and Polyllactosamines in Cancer Increased expression of galectins (especially galectin-3) associated with tumor progression and metastasisMechanism may involve interactions of galectins with polylactosamines on matrix proteins like laminin.Polylactosamines also expressed on cancer mucins and enriched on 1-6 branched glycans of tumor N-glycansThus, galectin-polyllactosamine interactions could mediate homotypic adhesion of carcinoma cellsRemains to be shown exactly how interactions of galectins & polyllactosamine alter cancer biology
39 Altered Expression ABO Blood Group Structures in Cancer Loss of normal AB blood group expression (accompanied by increased expression of H and Le y) associated with a poorer prognosis of carcinomas in several studiesReason for this correlation remains unknownRarely, a tumor may present an “illegal” blood group (i.e. expression of B blood group in an A-positive patient)Ggenetic basis for such a change remains unexplainedTumor regression noted in a few such cases, presumably mediated by naturally occurring endogenous antibodies
40 Changes in the Amount, Linkage and Type of Sialic Acids Most tumor cells show overall increase in cell surface sialic acid contentGreatest increase in metastatic tumorsIncreased Sia shown to reduce attachment of tumor cells to collagen type IV and fibronectinIncrease in Sia2-6Galb1-4GlcNAc (ST6Gal-I product) and Sia2-6GalNAc -O-Ser/Thr (Sialyl-Tn) in some tumors. Associated with poor prognosis.9-O-acetylated ganglioside GD3 in melanomas from a wide variety of species, ranging from humans to fishLoss of sialic acid 9-O-acetylation in Colon carcinomasBiological significance of most of these unknown
41 N-glycolylneuraminic Acid (Neu5Gc) in Human Tumors Neu5Gc differs from Neu5Ac by single oxygen atom, which is added by a specific hydroxylaseHumans are deficient in Hydroxylase and in Neu5GcHumans mount immune response to Neu5Gc in infused animal serumReports of aberrant Neu5Gc expression in human tumorsIs there an alternate pathway for synthesis of Neu5Gc?Or does it come from dietary sources?Neu5Gc accumulation may explain why some patients with cancer spontaneously develop "Hanganutziu-Deicher" “serum-sickness-like” antibodies that are directed against Neu5Gc-containing gangliosides.