Essentials of Glycobiology Ajit Varki Lecture 35

1. Essentials of Glycobiology Ajit VarkiLecture 35 Changes in Glycosylation in Cancer

2. INHERITED GENOMIC DNA ABNORMALITIES DNA DAMAGE CAUSED BY EXTERNAL AGENTS IMMORTALIZATION OF GROWTH POTENTIAL ABNORMAL ONCOGENE EXPRESSION CAUSING INCREASED GROWTH LOSS OF TUMOR SUPPRESSOR GENESTHAT NORMALLY CONTROL SUCH CELLS ACCUMULATION OF GENETIC ABNORMALITIES DEVELOPMENT OF GENETIC HETEROGENEITY, ALLLOWING TUMOR SPREAD (METASTASIS) METASTASIS IS NOW THE MAIN CAUSE OF DEATH IN HUMANS WITH CANCER CLINICALLY RELEVANT CANCER INVOLVES A MULTISTEP PROCESS

3. Clonal Expansion and Growth Angiogenesis Invasion of Basement Membrane Passage through Extracellullar Matrix Intravasation Tumor Cell interactions with Blood cells Adhesion to endothelium Invasion of basement membrane Extravasation Growth and Angiogenesis

4. 1950s: Enhanced binding of certain plant lectins (e.g., wheat germ agglutinin) to animal tumor cells 1960s: In vitro transformation of cells frequently accompanied by increases in overall size of metabolically labelled glycopeptides. 1970-80s: Search for “magic bullet” monoclonal antibodies against cancer. Many “tumor-specific” antibodies directed against carbohydrate epitopes, especially on glycosphingolipids. 1980-90s: These epitopes are actually “oncofetal antigens” - also expressed in embryonic tissues, and in a few normal adult cell types Historical Background

5. 1970-90s: 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! Historical Background (Continued)

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. Loss of expression of certain structures Excessive expression of certain structures Persistence of incomplete or truncated structures Accumulation of precursors Appearance 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. General Ways In Which Glycosylation Can Be Altered in Malignant Cells 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 mucins • Glycosphingolipids: 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 Proteoglycans • GPI Anchors - losses in some leukemias.

9. Changes in Shared Outer Structures • Increased outer chain alpha1-3(4) Fucosylation, generating Selectin ligands • Increased Expression of Polylactosamines (Galectin Ligands) • Altered Expression of ABO Blood Group Structures • Increase in overall Sialic Acid content • Enhanced expression of some Sialic acid linkages • Alterations in Types of Sialic Acids

10. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS Ac GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan O-LINKED CHAIN P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

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. Transcriptionally increased expression of GNT-V induced by viral and chemical carcinogenesis Cell lines with increased GNT-V expression show increased frequency of metastasis Spontaneous revertants for enzyme expression lose metastatic phenotype Clinical specimens of some tumors show increased staining with the lectin L-PHA, which preferentially recognizes 1-6 branched N-glycans Increased GlcNAc Transferase-V mediated 1-6 Branching of N-glycans

14. Transfection of GNT-V cDNA into cells causes: Visually obvious transformed phenotype Enhanced colony formation in agar Increased cell spreading Enhanced invasiveness through membranes Tumorigenic behavior in previously non-tumorigenic cells. Increased GlcNAc Transferase-V mediated 1-6 Branching of N-glycans By conventional criteria, these are characteristics of a true oncogene!

15. Mgat5 (-/-) mice lack GlcNAcT-V products and appear fertile/normal Altered proliferation in lymphocytes and epithelium Mammary tumor growth and metastases induced by the polyomavirus middle T oncogene considerably decreased GNT-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 Suppression of Tumor growth and metastasis in (Mgat5) GlcNAcT-V-deficient mice

16. Precise mechanism(s) of biological outcomes unknown Increased 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. Increased 1-6 Branching of N-glycans in Cancer

17. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS Ac GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan O-LINKED CHAIN P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

18. Many "tumor-specific" monoclonal antibodies raised against tumors recognize glycosphingolipids Some highly enriched in specific types of tumors e.g., Gb3/CD77 in Burkitt’s lymphoma and GD3 in melanomas Some tumors (particularly melanoma and neuroblastoma) synthesize very high levels of gangliosides Some, e.g., GD2 and GM2 not found at high levels in extraneural cells - targets for immunotherapy In vitro studies suggest that some gangliosides can affect growth control Large quantities of gangliosides "shed" by some tumors have strong immunosuppressive effects Altered Expression/Shedding of Glycosphingolipids

19. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS Ac GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan O-LINKED CHAIN P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

20. Altered sugar chains CANCER MUCINS IN NORMAL AND MALIGNANT EPITHELIUM Mucins NORMAL BASEMENT MEMBRANE

21. Mucins are major carriers of altered glycosylation Mucin expression correlates with metastatic potential in some carcinomas Shed Mucins in body fluids have diagnostic value Mucins can block adhesion by cytolytic cells, e.g., NK cells. Mucins can show incomplete O-glycosylation Mucins with Altered O-Glycosylation in Cancer

22. Correlation between T and Tn expression, spontaneous antibodies directed against them, and prognosis The most extreme form of underglycosylation results in expression of "naked" mucin polypeptides Clinical trials underway to provoke/enhance these immune responses by injecting patients with peptide antigens, sometimes bearing multiple copies Tn of Sialyl-Tn Early results promising Truncated O-glycans on Mucins in Cancer

23. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS Ac GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan O-LINKED CHAIN P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

24. Epithelial tumors surrounded by stroma enriched in HA. Corresponding normal epithelia give very low signal for HA Extent of stromal HA accumulation -a strong, independent, negative predictor of survival in many cancers Three major molecular characteristics of HA contribute to normal and tumor cell behavior Unique hydrodynamic properties Interactions with HA-binding “hyaladherins” in the assembly of pericellular and extracellular matrices. Effects on cell signaling and behavior. Hyaluronan (HA) in Cancer

25. Extracellular matrix surrounding proliferating and migrating cells in embryonic development, regeneration, healing, cancer and vascular disease is highly enriched in HA Simple 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 rounding HA 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 rearrangements being deposited in cytoplasm? Several intracellular hyaladherins, e.g. Cdc37, IHABP4, an intracellular form of RHAMM are known Hyaluronan in Cancer

26. 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 angiogenesis Soluble CD44 induces G1 arrest or apoptosis in tumor cells and inhibition of MMP-mediated invasion. Hyaluronan in Cancer - evidence in animal models

27. 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 vivo Overexpression of RHAMM leads to enhanced tumor growth and metastasis Hyaluronan in Cancer - evidence in animal models

28. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS Ac GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan O-LINKED CHAIN P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

29. 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 Sulfated Glycosaminoglycans in Cancer

30. Multiple Hereditary Exostosis • Autosomal dominant trait • Bony outgrowths (exostoses) usually located next to epiphyseal growth plate • Skeletal deformities such as shortened or curved limbs, reduced stature • Pain caused by muscle and nerve compression • 0.5 - 2% predilection for progression to malignant chondrosarcoma, i.e., a “tumor-suppressor gene”

31. Patients with Multiple Hereditary Exostosis have mutations in EXT1 and EXT2 The heparan sulfate copolymerase consists of an heterooligomer of EXT1 and EXT2 GlcA and GlcNAc transferase active sites of the copolymerase reside in separate subdomains Several mutations in EXT1 and EXT2 can result in deficient heparan sulfate synthesis Heterozygous state produce hereditary exostosis Note: homozygous null state is lethal and mice, and not seen in living humans Multiple Hereditary Exostosis

32. = Sialic acid Common Outer Chains Shared by Different Classes of Glycans S N-LINKED CHAIN O N Ser/Thr Asn O-LINKED CHAIN Secreted Protein GLYCOSPHINGOLIPID S O N Membrane Protein Ser/Thr Asn OUTSIDE CELL MEMBRANE INSIDE

33. from Nakamori et.al, 1997 Prognostic significance of Sialyl Lewisx expression in patients with colon adenocarcinoma Siaa2-3Galb1-4GlcNAcb1- 3 1 a Fuc SLex expression also associated with poor prognosis in carcinomas of Breast, Lung, Pancreas, Bladder, Prostate, Biliary tree and Ovary DO SELECTINS PLAY IN ROLE IN ADENOCARCINOMA PROGRESSION?

34. L L L L L P P P P P E E E L-selectin P-selectin E-selectin Tissue Factor? Resting platelet Thrombin Interactions between Carcinoma Mucins and Selectins Leukocyte APt Activated Carcinoma platelet cell APt Leukocyte Soluble Mucin Membrane- bound Mucin Activated Endothelium Selectin Binding sites

35. Effects of Selectin Deficiencies on the Metastatic Progression of GFP-expressing MC-38 Mouse Adenocarcinoma in Syngeneic Mice • P- and L-selectin deficiency are additive • P-selectin deficiency works by preventing tumor cell interactions with blood platelets • Mechanism(s) of metastasis reduction by L-selectin deficiency unknown • E-selectin may also play a role

36. Heparin (Others) Control (Others) Heparin Heparin (Cancer) Control Control (Cancer) Perioperative Heparin Therapy Reduces Late Deaths from Metastatic Cancer Kakkar et al. Int.J.Oncol. 6:885,1995. 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-selectin • Inhibition of the Clotting Cascade • Interactions with Integrins • Binding up of Growth factors • Inhibition of Angiogenesis • Inhibition of Heparanases • Alterations of Protease actions • Lots of clinical heparin (standardized for anticoagulation) may vary in the other actions • Very few studies show a detrimental consequence of heparin in murine or human cancer

38. Increased expression of galectins (especially galectin-3) associated with tumor progression and metastasis Mechanism 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-glycans Thus, galectin-polyllactosamine interactions could mediate homotypic adhesion of carcinoma cells Remains to be shown exactly how interactions of galectins & polyllactosamine alter cancer biology Galectins and Polyllactosamines in Cancer

39. 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 studies Reason for this correlation remains unknown Rarely, 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 unexplained Tumor regression noted in a few such cases, presumably mediated by naturally occurring endogenous antibodies Altered Expression ABO Blood Group Structures in Cancer

40. Most tumor cells show overall increase in cell surface sialic acid content Greatest increase in metastatic tumors Increased Sia shown to reduce attachment of tumor cells to collagen type IV and fibronectin Increase in Sia2-6Galb1-4GlcNAc (ST6Gal-I product) and Sia2-6GalNAc  -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 fish Loss of sialic acid 9-O-acetylation in Colon carcinomas Biological significance of most of these unknown Changes in the Amount, Linkage and Type of Sialic Acids

41. Neu5Gc differs from Neu5Ac by single oxygen atom, which is added by a specific hydroxylase Humans are deficient in Hydroxylase and in Neu5Gc Humans mount immune response to Neu5Gc in infused animal serum Reports of aberrant Neu5Gc expression in human tumors Is 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. N-glycolylneuraminic Acid (Neu5Gc) in Human Tumors