1. Essentials of Glycobiology Ajit Varki Lecture 35
Changes in Glycosylation in Cancer

2. CLINICALLY RELEVANT CANCER INVOLVES A MULTISTEP PROCESS
INHERITED GENOMIC DNA ABNORMALITIES
DNA DAMAGE CAUSED BY EXTERNAL AGENTS
IMMORTALIZATION OF GROWTH POTENTIAL
ABNORMAL ONCOGENE EXPRESSION CAUSING INCREASED GROWTH
LOSS OF TUMOR SUPPRESSOR GENES THAT 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

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. Historical Background
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.
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 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.
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. Major Glycan Classes in Animal Cells
HYALURONAN
GLYCOSAMINO-
GLYCANS
HEPARAN SULFATE
CHONDROITIN
SULFATE
Major Glycan Classes in Animal Cells P S S S
Ser-O- S S S S S
-O-Ser NS NS
Proteoglycan Ac
N-LINKED CHAINS
O-LINKED
CHAIN
GLYCOPHOSPHO-
LIPID
ANCHOR P S
Etn P O N N
Ser/Thr
Asn
Asn NH 2
GLYCOSPHINGOLIPID
Glycoprotein
INOSITOL Ac
OUTSIDE P
Sialic Acids
INSIDE
O-LINKED GlcNAc 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. Increased GlcNAc Transferase-V mediated 1-6 Branching of N-glycans
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

14. Increased GlcNAc Transferase-V mediated 1-6 Branching of N-glycans
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.
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/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

16. Increased 1-6 Branching of N-glycans in Cancer
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.

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

18. Altered Expression/Shedding of Glycosphingolipids
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

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

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

21. Mucins with Altered O-Glycosylation in Cancer
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

22. Truncated O-glycans on Mucins in Cancer
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

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

24. Hyaluronan (HA) in Cancer
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.

25. Hyaluronan in Cancer
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

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 angiogenesis
Soluble 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 vivo
Overexpression of RHAMM leads to enhanced tumor growth and metastasis

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

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 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
% 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 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

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

33. Prognostic significance of Sialyl Lewisx expression in patients with colon adenocarcinoma
from Nakamori et.al, 1997
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. Interactions between Carcinoma Mucins and Selectins
Tissue Factor?
Resting platelet
Thrombin
APt
Leukocyte
Activated
Carcinoma
L-selectin
P-selectin
platelet
cell L P
APt
Leukocyte
Soluble
Mucin
Membrane-
bound Mucin E
E-selectin
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. Perioperative Heparin Therapy Reduces Late Deaths from Metastatic Cancer
Control
Heparin (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-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. Galectins and Polyllactosamines in Cancer
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

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 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

40. Changes in the Amount, Linkage and Type of Sialic Acids
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

41. N-glycolylneuraminic Acid (Neu5Gc) in Human Tumors
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.