Essentials of Glycobiology Ajit Varki Lecture 35 Changes in Glycosylation in Cancer
Dec 15, 2015
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 β4
β3
β4
α4
α4GNT-V
Selectins?
α3α6
β4
β4 β4 α3/6
GNT-III
Galectins?
α3/6
α3/6 α3/6
Altered N-Linked Glycosylation in Tumor cells due to Overexpression of GNT-V
α3
α4
β6
β4 β4
β4β4 β4
β3
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
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 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
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
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
TnAntigen
-Sialyl Tn
TAntigen
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
TnAntigen
-Sialyl Tn
TAntigen
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
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
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
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
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID
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.