Essentials of Glycobiology Ajit Varki Lecture 35

Essentials of Glycobiology

Ajit Varki

Lecture 35

Changes in Glycosylation in Cancer

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

Clonal Expansion and GrowthAngiogenesis

Invasion of Basement Membrane

Passage through Extracellullar Matrix

Intravasation

Tumor Cell interactions with Blood cells

Adhesion to endotheliumInvasion of basement membrane

ExtravasationGrowth and Angiogenesis

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.

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 (Continued) 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!

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.

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

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.

Changes in Core Structures and Core Proteins

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

Changes in Shared Outer Structures

Major Glycan

Classes in Animal Cells

OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

S

2

P

GlycoproteinGlycoprotein

ProteoglycanProteoglycan

GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID

ANCHORANCHOR

O-LINKED O-LINKED CHAINCHAIN

Ac

N-LINKED CHAINSN-LINKED CHAINS

HYALURONANHYALURONAN

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE

CHONDROITINCHONDROITIN SULFATESULFATE

Sialic AcidsSialic Acids

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

O-LINKED GlcNAcO-LINKED GlcNAc

Altered N-Linked Glycosylation in Tumor cells due to Overexpression of GNT-V

α3

α4

β6

β4 β4

β4β4 β4β3

β4

α4

α4GNT-V

Selectins?

α3α6

β4

β4 β4 α3/6

GNT-III

Galectins?

α3/6

α3/6 α3/6

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

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 agar Increased cell spreadingEnhanced invasiveness through membranesTumorigenic behavior in previously non-

tumorigenic cells.

By conventional criteria, these are characteristics of a true oncogene!

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

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.

Major Glycan


OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

S

2

P




ANCHORANCHOR


Ac








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

Major Glycan


OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

S

2

P




ANCHORANCHOR


Ac








MUCINS IN NORMAL AND MALIGNANT EPITHELIUM

BASEMENTMEMBRANE

NORMAL

Altered sugar chains

CANCERMucins

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

Ser/Thr

ß3

α6

UDP-

Tn Antigen

Siαlyl-Tn

T Antigen

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

Ser/Thr

ß3

α6

UDP-

Tn Antigen

Siαlyl-Tn

T Antigen

Major Glycan


OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

S

2

P




ANCHORANCHOR


Ac








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.

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

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.

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

Major Glycan


OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

S

2

P




ANCHORANCHOR


Ac








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

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”

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

Common Outer Chains Shared by Different Classes of Glycans

OSer/Thr

NAsn

N-LINKED CHAINN-LINKED CHAIN

O-LINKED CHAINO-LINKED CHAIN


OUTSIDE

INSIDE

S

CELLMEMBRANE

Membrane ProteinMembrane Protein

OSer/Thr

NAsn

S

= Sialic acid

Secreted ProteinSecreted Protein

from Nakamori et.al, 1997

Prognostic significance of Sialyl Lewisx expression in patients with colon adenocarcinoma

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?

Siaα2-33Gal1-44GlcNAc1-331αFuc

Interactions between

Carcinoma Mucins and

Selectins

Membrane-bound Mucin

Activated Endothelium

APt

Soluble

Mucin

Carcinoma cell

E-selectin

Activated platelet

Resting platelet

P-selectin

Leukocyte

LeukocyteAPt

Selectin Binding sites

Tissue Factor?

L-selectin

Thrombin

L

L

L

L

L

P

P

P P

P E

EE

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

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

Heparin

Control

Heparin (Others)

Control (Others)

Heparin (Cancer)

Control (Cancer)

Diverse Effects of Heparin in Cancer

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

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

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

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

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 Siaα2-6Gal1-4GlcNAc (ST6Gal-I product)

and Siaα2-6GalNAc -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

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.

Essentials of Glycobiology Ajit Varki Lecture 35

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