Você está na página 1de 12

REVIEWS

GLYCANS IN CANCER AND INFLAMMATION POTENTIAL FOR THERAPEUTICS AND DIAGNOSTICS


Danielle H. Dube* and Carolyn R. Bertozzi*,,
Abstract | Changes in glycosylation are often a hallmark of disease states. For example, cancer cells frequently display glycans at different levels or with fundamentally different structures than those observed on normal cells. This phenomenon was first described in the early 1970s, but the molecular details underlying such transformations were poorly understood. In the past decade advances in genomics, proteomics and mass spectrometry have enabled the association of specific glycan structures with disease states. In some cases, the functional significance of disease-associated changes in glycosylation has been revealed. This review highlights changes in glycosylation associated with cancer and chronic inflammation and new therapeutic and diagnostic strategies that are based on the underlying glycobiology.
The treatment of diseases such as cancer and inflammation is extremely challenging because the pathology involves dysregulation of endogenous and often essential cellular processes. Effective therapies typically capitalize on differences between diseased and healthy tissues that can be targeted with drugs. Many cancer drugs, for example, target the cell cycle (for example, DNA replication and cytoskeletal formation). More recently, signalling pathways have become the focus of new anticancer drugs (for example, kinases and phosphatases)1,2. Inflammation therapies focus on suppressing the immune system (for example, antagonists of tumour-necrosis factor- (F), steroids)3. Though combination therapies show improved selectivity, these therapies suffer from side effects that result from imperfect selectivity for disease tissue. The availability of novel molecular targets that distinguish diseased from healthy cells could vastly amplify therapeutic opportunities. An ongoing challenge is therefore to identify other diseaseassociated changes in cellular physiology that might be harnessed for more-selective treatment and diagnosis. Glycobiologists have known for decades that the structures of glycans, which decorate all eukaryotic cell surfaces, change with the onset of cancer4 and inflammation57. The glycosylation pattern of a cell is therefore a code for cellular physiology. An understanding of this code at both molecular and functional levels is starting to emerge. Although glycobiologists are still at the early stages of deciphering this code, their results have already inspired novel methods to detect glycans characteristic of disease conditions, to prevent disease glycan formation and to destroy cells that display them. Here we provide an overview of the link between glycan structures and disease progression, with a particular focus on cancer and chronic inflammation. We also highlight opportunities for therapeutic and diagnostic approaches that are based on the underlying glycobiology. This burgeoning field should provide considerable fuel for new academic and industrial enterprises.
Glycobiology

Departments of *Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. Correspondence to C.R.B. e-mail: crb@berkeley.edu
doi:10.1038/nrd1751

Although it has long been appreciated that glycan expression changes with cellular condition, progress toward delineating the molecular basis of glycan function has been rather slow relative to comparable studies of proteins and nucleic acids. This slow progress is partly due to the fact that the biosynthesis of glycans,

NATURE REVIEWS | DRUG DISCOVERY

VOLUME 4 | JUNE 2005 | 477

2005 Nature Publishing Group

REVIEWS
unlike other biopolymers, is not template-driven. In mammals, glycans are constructed from nine monosaccharide building blocks that can be connected to one another through glycosidic linkages in myriad combinations. Glycans are formed on their protein or lipid scaffolds by GLYCOSYLTRANSFERASES and GLYCOSIDASES as they traffic through the secretory pathway. The combined action of these enzymes in the secretory pathway leads to a diverse array of glycan structures. The major classes of glycan structures are shown in BOX 1. N-linked glycans are attached to an asparagine residue of a protein carrier, O-linked glycans are attached to serine or threonine residues, and glycosaminoglycans (GAGs) are attached to serine residues of proteoglycan molecules. By contrast, glycolipids have a lipid carrier, such as sphingolipid. Glycan structures can vary from highly branched and complex glycans (for example, N-linked and O-linked glycans, and glycolipids) to linear glycans (for example, proteoglycans). Due to the large number of possible structures, the information content of glycans is enormous. Cracking the glycan code as it relates to disease states has been a major focus of glycobiologists during the past several decades8.
Description of tumour-associated glycans

Box 1 | Glycobiology primer The combined action of glycosyltransferases and glycosidases in the secretory pathway leads to a diverse array of glycan structures. The major classes of glycan structures are shown. N-linked glycans are attached to an asparagine residue of a protein carrier, O-linked glycans are attached to serine or threonine residues, and glycosaminoglycans (GAGs) are attached to serine residues. In contrast to glycoproteins, glycolipids have a lipid carrier, such as the sphingolipid depicted in the diagram. Glycan structures can vary from highly branched and complex glycans (for example, N-linked and O-linked glycoproteins, or glycolipids) to linear glycans (for example, GAGs).
HO O HO O AcHN HO HO HO O O

O HO O HO HO HO O HO HO O NHAc H3C OH OH HO AcHN HO OH OH HO AcHN HO O O CO2 O OH CO2 O OH OH O O OH OH O OH O O HO

OH O NHAc O HO

OH O NHAc NH

H N N H O

N-linked glycan

OH NHAc O O O

HO OH OH O OH

O HO O O AcHN O H (CH3) H N N H O

Mucin-type O-linked glycan

OSO3
O2C

OH O
O2C

OH O O OH

OH O

OH O

OH O O

O HO

O O OH

O HO

O HO

AcHN

n n = 2060

OH

OH HO

O H N O

Glycosaminoglycan (GAG)
OH O O OH O OH O OH HN O HO OH O
12 16

N H

OH O

O CH3

AcHN OH OH HO AcHN HO O CO2 O

CH3

OH

Glycolipid

Altered glycosylation patterns are a hallmark of the tumour phenotype. This phenomenon was first described by Meezan et al. in 1969 with the demonstration that healthy fibroblasts have smaller membrane glycoproteins than their transformed counterparts4. This finding was later corroborated with histological evidence that LECTINS show differential binding to healthy compared with malignant tissue5,9. More recently, cancer-associated cell-surface glycans have been directly characterized using specific monoclonal antibodies and mass spectrometry10, further illuminating the molecular changes that occur upon malignant transformation11,12. We now know that changes in glycosylation include both the under- and overexpression of naturally-occurring glycans, as well as NEOEXPRESSION of glycans normally restricted to embryonic tissues. These structures most often arise from changes in the expression levels of glycosyltransferases in the Golgi compartment of cancerous cells. Changes in glycosyltransferase levels can lead to modifications in the core structure of N-linked and O-linked glycans. One of the most common changes is an increase in the size and branching of N-linked glycans. This increased branching is often attributed to increased activity of N-acetylglucosaminyltransferase V (GlcNAc-TV, also known as or MGAT5; the enzyme that leads to 1,6GlcNAc branching)13. The increased branching creates additional sites for terminal sialic acid residues, which, in conjunction with a corresponding upregulation of sialyltransferases, ultimately leads to an increase in global sialylation14. In addition to changes in the core structures of glycans, altered terminal structures are also associated with malignancy. Glycosyltransferases (for example, sialyltransferases and fucosyltransferases) involved in linking terminating residues on glycans tend to be overexpressed in tumour tissue. The increase in activity of these glycosyltransferases in turn leads to the overexpression of certain terminal glycans. Examples of terminal glycan epitopes commonly found on transformed cells include sialyl Lewis x (sLex), sialyl Tn (sTn), Globo H, Lewis y (Ley) and polysialic acid (PSA) (FIG. 1)1518. Many of these epitopes are observed

478 | JUNE 2005

| VOLUME 4
2005 Nature Publishing Group

www.nature.com/reviews/drugdisc

REVIEWS

OH OH HO AcHN HO O

CO2 O

OH

OH O OH O

AcHN OR O HO O OH O HO AcHN

OH OH O HO

CO2 O

OH

OH O OH O O

OH O OR

NHAc O OH

H3C HO OH

H3C HO OH

Sialyl Lewis a (sLea)

Sialyl Lewis x (sLex)


OH O O O O HO O OH O HO O OH OH O OH OR OH OH O

OH OH HO AcHN HO HO O

CO2 OH

OH O O H3C O OH HO

OH O

OH

NHAc

OH

HO

AcHN OR

HO

Sialyl Tn (sTn)
OH O HO O H3C HO OH O HO OH HO HO AcHN
O C 2

Globo H
OH O NHAc O OH OH O O O OH O O HO OH OH HN O O
12 16

OH OH HO AcHN OR OH O

CH3

CH3

O OH

OH

Tn
OH OH HO AcHN HO O HO AcHN HO CO2

Fucosyl GM1

n
O OH O HO AcHN HO CO2 O OH O CO2 HO OR H3C OH O O OH

OH O OH O O

OH O OH O

OH

OH O OH OR

OH H3C HO OH

OH

(n = 1050)

Polysialic acid (PSA)

Lewis y (Ley)

Figure 1 | Cancer-associated glycans. Altered glycosylation patterns are a hallmark of the tumour phenotype, consisting of both the under- and overexpression (for example, sialyl Lewis x (sLex)) of naturally-occurring glycans as well as the presentation of glycans normally restricted to expression during embryonic development (for example, polysialic acid (PSA)). The oligosaccharide epitopes depicted here are a small subset of glycans that are found on malignant cells. Glycans presented on a cells surface act as a code to convey information about the physiological state the cell. For example, high levels of the capping monosaccharide sialic acid suggest a poor prognosis (high metastatic ability) in many types of cancers.
GLYCOSYLTRANSFERASES

Enzymes that form glycosidic bonds between monosaccharide units.


GLYCOSIDASES

Enzymes that cleave glycosidic bonds.


LECTINS

Carbohydrate-binding proteins.
NEOEXPRESSION

Expression on adult tissues, when expression is normally restricted to embryonic tissues.


GANGLIOSIDE

A glycosphingolipid-containing sialic acid.

in malignant tissues throughout the body, including the brain, breast, colon and prostate TABLE 119. Another common feature of tumours is the overproduction of certain glycoproteins and glycolipids. For example, epithelial tumours often overproduce mucin glycoproteins, which are characterized by dense clusters of O-linked glycans. Mucins are used as diagnostic markers of cancer and can also function as scaffolds for most of the above-listed cancerassociated epitopes20. Additionally, cancer tissues can display an increase in GANGLIOSIDE expression. For example, complex gangliosides (for example, GD2, GD3 and fucosyl GM1) (FIG. 1) are found at elevated levels in small-cell lung carcinomas, neuroblastomas and melanomas16,21. Although gross changes in glycosylation of tumour tissues are apparent, no single

change seems to distinctly differentiate normal and malignant cells. Instead, each type of malignant tissue is characterized by a distinct set of changes in glycan expression TABLE 122,23.
Carbohydrate-based anticancer vaccines

Because cancer cell glycans differ from those found on their healthy counterparts, it might be possible to recruit the immune system to target cancer cells on the basis of their altered glycosylation. Although atypical glycans can render cancer cells mildly antigenic (that is, capable of eliciting specific antibodies), they are rarely immunogenic (that is, the antibodies are not capable of recruiting immune effector functions to kill the cells)24. Many tumour-associated glycans have an embryonic origin or are expressed at low levels in normal tissue

NATURE REVIEWS | DRUG DISCOVERY

VOLUME 4 | JUNE 2005 | 479

2005 Nature Publishing Group

REVIEWS

Table 1 | Common expression patterns of cancer glycans on malignant tissues*


Cancer glycan Ovary sLe
x

Malignant tissue Pancreas X X X X X X X X X X X X Blood Breast X X X X X X X Colon X X X X X X X X X X X X X X X X X X X X X X X X X X X X Brain Prostate Skin Lung X X X

sLea sTn TF Ley Globo H PSA GD2 GD3 Fucosyl GM1 GM2
*Based on REFS 22,23.

and at elevated levels on tumours. Consequently, the glycans can be perceived as self by the human immune system, in which case B cells expressing high-affinity antibodies for these structures would have been eliminated during development25,26. For this reason, many attempts at generating anticancer vaccines have focused on breaking immune self-tolerance for tumour-associated glycans27,28. Danishefsky, Livingston and co-workers have prepared an array of carbohydrate-based anticancer vaccines by linking multiple copies of synthetic tumour-associated glycans to the immunogenic carrier protein keyhole limpet haemocyanin (KLH)27. This foreign protein provides peptide antigens that are required for T-cell help and a full-blown immune response. When administered to mice and/or humans, many of these glycoconjugate vaccines elicited antibody- and cell-mediated immune responses against the glycan27,2934 (FIG. 2). Several glycan-based vaccines are presently undergoing clinical evaluation and have shown some promise27,2935 TABLE 2. In a Phase III clinical trial for metastatic breast cancer, the sTnKLH conjugate Theratope (Biomira) failed to meet the endpoints of time-to-disease progression and overall survival. However, patients treated with Theratope in conjunction with hormone therapy had improved survival rates with a time-to-disease progression of 8.3 months compared with 5.8 months for those on hormone therapy alone36. This modest clinical efficacy could be due to the fact that Theratope elicits a B-cell-mediated immune response but does not seem to trigger a T-cell-mediated immune response36. Successful tumour immunotherapy might require the induction of cytotoxic T lymphocytes (CTLs) in addition to antibodies. There are strategies currently being pursued to recruit T-cell help, including mimicking the cancer cell surface by displaying vaccine glycans in a multivalent context, chemically modifying cancer glycans to make them more foreign, and actively

recruiting T-cell help by exploiting the naturally abundant human anti-Gal antibody. Each of these glycan-based vaccine strategies is discussed below. Each type of malignant tissue is characterized by a distinct set of changes in glycan expression TABLE 1. A vaccine that targets several cancer-associated glycans should, in principle, lead to a stronger and more specific immune response than one that targets a single cancer glycan29,37. In an effort to mirror the heterogeneous nature of cancer-cell-surface glycans, Danishefsky and co-workers have prepared multiantigenic vaccines that contain several different glycan structures29,38. For example, a tri-antigenic vaccine containing Globo H, Ley and Tn has been shown in animal models to elicit an immune response against each oligosaccharide antigen29. In this preclinical study, antibodies that recruit T-cell help (immunoglobulin G (IgG) isotype) were generated against each glycan antigen. This promising result suggests that a multiantigenic approach might be successful at recruiting both humoral and T-cell mediated immune responses against tumours in human patients. Chemical modification of monosaccharide structures can augment the immunogenicity of glycanbased vaccines28,33. Chemically modified sialic acid residues with unnatural N-acyl side chains, such as N-propanoyl33,3941, N-butanoyl4042, N-phenylacetyl40,41 and N-levulinoyl43, have been incorporated into KLH conjugates40,41 and are more immunogenic than the corresponding natural sialic acid vaccines33,40,41 (FIG. 2). N-propanoyl, N-butanoyl and N-phenylacetyl sialic acid derivatives conjugated to KLH produced a robust immune response in mice. These derivatives elicited IgG antibody production, which recruits T-cell help. By contrast, the natural N-acetyl-sialic-acidKLH conjugate was essentially non-immunogenic40. Similarly, Livingston and co-workers conducted a preliminary clinical trial in which small-cell lung carcinoma patients were vaccinated with polysialic acid (PSA) KLH or N-propionylated PSAKLH (PrPSAKLH)

480 | JUNE 2005

| VOLUME 4
2005 Nature Publishing Group

www.nature.com/reviews/drugdisc

REVIEWS

a
PSA PSA PSAKLH conjugate PSA PSA

PSA

PSA

Mild immune response

b
PSA PSA PrPSAKLH conjugate PSA PSA

PSA

PSA

Stronger immune response

c
PSA PSA ManNProp
O HO

Pr-PSA Pr-PSA

Anti-PrPSA

Pr-PSA

Pr-PSA

PSA

HO HO

HN

CH3 O OH

Pr-PSA

Pr-PSA

Prevents metastasis Cancer cell Normal cell

Figure 2 | Carbohydrate-based anticancer vaccines. Upon stimulation with carbohydrate-based anticancer vaccines, the immune system can be stimulated to recognize cells presenting cancer glycans. a | For example, a vaccine containing the polysialic acid (PSA) epitope conjugated to the immunogenic carrier protein keyhole limpet haemocyanin (KLH) (PSAKLH) raised a mild immune response in animals33. b | Recent reports have found that the chemically modified (unnatural) PrPSAKLH conjugate raised a stronger immune response in animals than the natural PSAKLH conjugate33. c | Finally, a passive immunization strategy in which mice were treated with N-propanoylmannosamine (ManNProp), a biosynthetic precursor of N-propionylated PSA, and a monoclonal antibody raised against synthetic N-propionylated PSA (anti-PrPSA), showed success in preventing metastasis of PSA-positive cells in mice39.

PASSIVE IMMUNIZATION

Treatment with an antibody, not actively recruiting the animals immune response.

conjugates. The modified PrPSAKLH conjugate elicited specific antibodies that crossreacted with PSA, whereas the natural PSA conjugate did not (FIG. 2)33. Chemically modified sialic acid conjugates have also shown success in PASSIVE IMMUNIZATION in animal models. Jennings and co-workers demonstrated that administration of a monoclonal antibody raised against a synthetic PrPSAKLH conjugate prevented metastasis of a leukaemic cell line when the mice were treated with N-propanoylmannosamine, a biosynthetic precursor of N-propionylated PSA (FIG. 2)39. More recently, the same group applied this passive immunization strategy to melanomas expressing the sialylated glycolipid GD342. T-cell-mediated immune reactions against cancer glycans can be elicited using the natural human antiGal antibody as an endogenous adjuvant44. Although

cancer glycans are not normally detected by the human immune system, they can be processed and presented to T-cells if the cancer cells express another epitope on them that is detected by the immune system. Galili and co-workers have developed a vaccine strategy that takes advantage of the anti-Gal antibody, which constitutes ~1% of circulating IgG in humans. The anti-Gal antibody reacts specifically with the -Gal epitope (Gal1,3Gal1,4GlcNAcR), which is entirely absent from human tissues. This antibody is thought to originate from widespread exposure to bacteria that express similar glycans. The -Gal epitope can be enzymatically synthesized on human tumour cells by the use of recombinant -1,3-galactosyltransferase (1,3GT)45 or by infection of the tumour cells with adenovirus containing the gene encoding 1,3GT46. These -Gal epitope-expressing tumour cells or their
VOLUME 4 | JUNE 2005 | 481

NATURE REVIEWS | DRUG DISCOVERY

2005 Nature Publishing Group

REVIEWS

Table 2 | Carbohydrate-based anticancer vaccines under development


Indication Malignant melanoma Breast cancer Prostate cancer Prostate cancer Vaccine GMK (GM2 conjugate) (Progenics) Theratope (sTnKLH conjugate) (Biomira) Globo H conjugate (Optimer) Globo HKLH Multiple vaccine treatment strategy: glycosylated MUC1-32mer, GM2, Globo H, Tn(c), TF(c) and Ley Tn(c)KLH L-BLP25 (Biomira): liposomal uMUC1 peptide vaccine Fucosyl-GM1 PSAKLH PrPSAKLH LeyKLH
y

Clinical status Phase III Phase III Phase I Phase II and III Phase II

References/source 116 36 115 31,115,117 127

Prostate cancer Non-small-cell lung carcinoma Small-cell lung carcinoma Small-cell lung carcinoma Ovarian cancer

Phase I Phase II Phase I Phase I Phase I

38 www.biomira.com 118 33 119

KLH, keyhole limpet haemocyanin; Le , Lewis y; MUC1, mucin 1; PSA, polysialic acid; PrPSA, N-propionylated PSA.

membranes can then be used as a vaccine to elicit an immune response against tumour-associated antigens47. Finally, there are several examples of specific glycoproteins that undergo changes in glycosylation upon malignant transformation. These include mucin 1 (MUC1)48, MUC16 (also known as CA125)49, prostate-specific antigen50,51 and carcinoembryonic antigen (CEA)52. MUC1, a mucin-type glycoprotein containing numerous O-linked glycans, is found on healthy breast tissue and on breast cancer tissue, but it is aberrantly glycosylated in the cancerous tissue 20,48 . Truncation of the O -glycans in breast cancer leads to the appearance of novel carbohydrate epitopes, such as Thomas Friedrich (TF) and sialyl Tn antigens 17,48,53. These changes in glycosylation reflect both the upregulation of a sialyltransferase (ST3Gal1), which transfers sialic acid onto the nascent O-glycan, and the underexpression of the glycosyltransferase core II GlcNAc-TI, which transfers another sugar (GlcNAc) to these sites53,54. Vaccines based on underglycosylated MUC1 (uMUC1) are currently undergoing clinical evaluation with encouraging results5557 TABLE 2.
Tumour glycans: prospects for new drugs

that many factors other than glycosylation contribute to malignant transformation14. In many cases, the causal link between carbohydrates and malignant function is not clearly understood. Hypotheses range from protection of tumour cells from immune surveillance by their glycan coat to promotion of tumour-cell metastasis by glycan-mediated adhesion to distant sites14,20,62. However, in some cases the causal link is clear certain glycan structures (for example, 1,6GlcNAc-branching and sLex) contribute to oncogenic signalling63, transformation64 and metastasis13,62. Several approaches have been undertaken to assess the functional roles of tumour-associated glycans. In each case, cells with different glycosylation profiles have been selected or created, and their physiological behaviour has been compared. Cellular glycans have been structurally perturbed by treatment of cells with glycosylation inhibitors or glycosidases, or by altering cellular glycosyltransferase expression. Summarized below are examples of experiments that provide evidence that these tumour-associated changes in glycosylation are functionally relevant. Levels of sialic acid affect metastasis. Cells can be selected for alterations in cell-surface glycan expression using lectins65. Wheat germ agglutinin (WGA), a cytotoxic plant lectin that binds to sialic acid or -GlcNAc residues, was used to select WGA-resistant B16 melanoma cells. These WGA-resistant cells showed reduced cell-surface sialylation and metastatic activity relative to the parental B16 cell line66. This correlation suggested a role for sialic acid in promoting metastasis. This interpretation was further supported in an experiment by Tokuyama and co-workers. They addressed the functional role of sialic acid residues in tumour metastasis by stably transfecting BL6 melanoma cells with a sialidase, an enzyme that removes sialic acid residues from

MICROHETEROGENEITY

Combination of structures.

Carbohydrate-based anticancer vaccines can be effective irrespective of the functional significance of cancer-associated glycans. By contrast, drug development strategies based on preventing the formation of cancer glycans or interrupting their downstream interactions can only be effective if these glycans make a functional contribution to the disease. Despite mounting clinical evidence suggesting a correlation between changes in glycosylation and poor prognosis5861, dissecting the role of a specific glycan in the biology of a given tumour has proven to be extremely challenging14. Complications include the MICROHETEROGENEITY of glycosylation and the fact

482 | JUNE 2005

| VOLUME 4
2005 Nature Publishing Group

www.nature.com/reviews/drugdisc

REVIEWS

a
HO HO

OH O AcHN O

OH

OH O OH O HO AcHN

OH OH O HO

CO2 O

OH

OH O OH O O OH

OH O AcHN O

OH

OH O OH O

GTs

H3C HO OH

GlcNAc1,3-Gal

O-linked glycoprotein

O-linked glycoprotein

sLex epitope synthesized on glycan

b
HO HO

OH O AcHN O

OH

OH O OH O HO HO

OH O AcHN O

OH

OH O OH O

Truncated glycan O-linked glycoprotein GTs


OH O O OH O HO AcHN HO OH OH O CO2 O OH H3C HO OH O OH OH O O O OH OH O AcHN O OH OH OH O O

O-linked glycoprotein

OH HO HO O AcHN

OH

Disaccharide decoy

sLex synthesized on decoy

Figure 3 | Disaccharide decoys act as metabolic inhibitors of glycosylation. a | The disaccharide N-acetylglucosamine 1,3 galactose (GlcNAc1,3Gal) is synthesized on O-linked glycoproteins and is further elaborated by glycosyltransferases in the Golgi to form the sialyl Lewis x (sLex) epitope. b | In the presence of high concentrations of the disaccharide decoy, GlcNAc1,3Gal-O-napthalenemethanol, glycosyltransferases in the Golgi are diverted away from the normal glycoprotein substrate and instead form sLex on the decoy. Ultimately, cells treated with the disaccharide decoy have reduced levels of sLex and present truncated glycans on their cell surfaces.

glycans67. These transfected cells showed a significant reduction in lung colonization in a mouse model relative to the parental cell line. Increased 1,6GlcNAc branching of N-linked glycans transforms cells. The role of 1,6GlcNAc-branching of N-linked glycans in cancer has been extensively studied. Demetriou et al. addressed the functional role of the branching 1,6GlcNAc residue in malignant transformation by transfecting an immortalized lung epithelial cell line (Mv1Lu) with GlcNAc-TV, the glycosyltransferase that installs this residue on N-linked glycans. GlcNAc-TV-transfected cells formed solid tumours in mice, whereas untransfected cells did not. In addition, the transfected cells showed a loss of contact inhibition of growth and increased motility in culture64. These data provide strong evidence that upregulation of this enzyme can induce malignant change. Consistent with this hypothesis, disruption of GlcNAc-TV in mice reduced malignancy68. Dennis and co-workers showed that GlcNAc-TV-deficient mice have suppressed mammary tumour growth and metastasis relative to littermates expressing the glycosyltransferase68. Finally, Pierce and co-workers reported that GlcNAc-TV-expression levels regulate cadherin-associated homotypic cellcell adhesion and downstream signalling pathways63. Collectively, these data suggest a functional, rather than simply correlative, role for 1,6-GlcNAc branching in tumour formation.

SELECTINS

A family of carbohydratebinding proteins (lectins) expressed on activated platelets (P-selectin) and endothelial cells (P- and E-selectin).

sLexselectin interactions can promote metastasis. Tumour metastasis is facilitated by adhesion between tumour cells and either platelets in the bloodstream or remote endothelial cells69. A well-known example of a receptorligand binding interaction that initiates this adhesion involves the SELECTINS. These selectins bind to sLex expressed on tumour cells and are thought to contribute to tumour-cell migration to distant tissues70. Esko and co-workers used metabolic inhibitors, termed disaccharide decoys, to study the role of sLex in metastasis7174. The decoys act as competitive substrates of glycosyltransferases and ultimately lead to the production of truncated glycans on the cell surface 7174. A disaccharide precursor of sLex, GlcNAc 1,3Gal - O -napthalenemethanol, was shown to reduce levels of cell-surface sLe x (FIG. 3) on LS180 colon adenocarcinoma cells. Relative to untreated cells, these sLe x -deficient cells had decreased interactions with selectins, increased susceptibility to leukocyte-mediated lysis and a reduction of lung metastasis in a murine tumour model71. Additionally, no significant difference in metastasis was observed when decoy-treated and untreated cells were injected into a P-selectin-deficient mouse, supporting the hypothesis that the sLexP-selectin interaction participates in tumourcell dissemination7577. Experiments with metabolic decoys raise the possibility of using such compounds as antimetastatic drugs74.

NATURE REVIEWS | DRUG DISCOVERY

VOLUME 4 | JUNE 2005 | 483

2005 Nature Publishing Group

REVIEWS
co-workers found that heparin disrupted P-selectinmediated interactions of platelets with carcinoma cell-surface mucins75. These cell-surface mucins are sialylated and fucosylated and bear the P-selectinbinding epitopes sLex and sLea. These findings are particularly exciting because heparin is already an approved drug. Given the functional link between aberrant glycosylation and malignancy, therapeutics that block the formation of cancer-associated glycans or their downstream interactions should have an effect on tumour progression. A number of glycosylation inhibitors and receptor inhibitors for the treatment of cancer are currently under development in pharmaceutical companies (for example, GlycoDesign (GD39), Oxford Glycoscience (OGT719), GlycoGenesys (GCS-100)).
Diagnostics based on cancer glycans

Box 2 | Bioorthogonal chemistry in living systems Bioorthogonal chemistry offers a means to visualize metabolites such as sugars, lipids and cofactors non-invasively in vivo. To achieve this in practice requires a pair of functional groups that are mutually reactive in a physiological environment and, at the same time, inert to the biological milieu. One of these functional groups is appended to the biomolecule of interest (for example, a monosaccharide) and the other is attached to a reporter (for example, a magnetic resonance imaging (MRI) contrast reagent or an affinity purification tag). For bioorthogonal chemistry to proceed in a living animal, the reactants must display chemical selectivity and possess biological compatibility they must not produce harmful side effects nor experience metabolic breakdown on the timescale of the reaction. We reported a reaction that fulfils these criteria the Staudinger ligation between azides and a specific class of phosphines90. The azide is essentially unreactive with biological nucleophiles yet readily condenses with exogenously delivered phosphine reagents. If the phosphine is functionalized as shown, its reaction with azides forms a stable amide bond with concomitant release of nitrogen and methanol and conversion to the phosphine oxide (see reaction scheme). The Staudinger ligation is the only synthetic reaction known to function on cells within living animals87. The ability of the Staudinger ligation to probe glycosylation in vivo has significant implications for imaging disease-associated glycans. For example, the Staudinger ligation might provide the basis of new cancer and inflammation diagnostic strategies (FIG. 4). Furthermore, this chemistry might be useful for identifying targets for drug discovery. Many disease-associated antigens are still undefined. By chemically tagging glycans with affinity purification probes, disease-associated glycans and their scaffolds can be enriched and identified.
O OCH3

N2
+

OCH3

P Ph Ph

+ N3

P Ph Ph

Phosphine

Azide

Aza-ylide

CH3OH O N H P Ph Ph O O N

H2O
Ph

P Ph

Ligation product Biological molecule of interest (for example, monosaccharide) Reporter molecule (for example, radionuclide or MRI contrast reagent)

BIOORTHOGONAL FUNCTIONAL GROUP

A chemical moiety that reacts selectively with a reaction partner in a physiological environment yet is inert to the biological milieu.

In complementary work, Shirota et al. investigated the effects of an inhibitor of sLexselectin binding on metastasis78. They used GSC-150, which is an analogue of sLex that competitively binds to the selectins. Co-administration of mice with GSC-150 and the colon carcinoma cell line KM12-HX reduced metastasis to the liver compared with mice treated with the cells alone. This result suggested that the sLexselectin interaction is a contributing factor to metastasis and that selectin inhibitors might find use in cancer treatment. Interestingly, the clinically approved anticoagulant drug heparin inhibits tumour metastasis in experimental animal models79 and has shown beneficial effects in human clinical trials of colon cancer80. Varki and

Glycan changes that indicate malignancy can be used for diagnosis. Indeed, the commonly used CEA test for colon cancer monitors serum levels of an antibody specific for a cancer-associated glycan (sLea)52. Glycan analyses of other serum markers, such as CA12549 and prostate-specific antigen50,51, reveal distinct changes in glycosylation in ovarian and prostate tumour tissue, respectively. These studies suggest that specific changes in glycosylation could be useful as diagnostic tools. For many cancers, however, there are no serum markers available. Instead, the tissue must be analysed directly. Existing diagnostic methods used to monitor tumour-specific glycosylation require surgical biopsy followed by histological analysis with lectins or monoclonal antibodies. An interesting future direction in the field is to target aberrant glycosylation with probes for image contrast17. To date, the field of molecular imaging has focused on protein-based markers that are targeted with antibody conjugates, receptor ligands or enzyme substrates/inhibitors8183. So far there is only one report of indirectly imaging a change in glycosylation84,85. Underglycosylated MUC1 antigen (uMUC1) is highly overexpressed in a number of human epithelial cell adenocarcinomas and is an early marker of tumorigenesis48,53. In its underglycosylated state, this protein can be recognized by a specific peptide (termed EPPT1) derived from a monoclonal antibody17,84. Epenetos and co-workers developed an EPPT1-99mTc probe to monitor breast cancer in vivo84. More recently, Dai and co-workers synthesized an EPPT1 conjugate capable of magnetic resonance and optical imaging85. Experiments with this multimodal probe in uMUC1positive murine tumour models showed specific accumulation of the probe in the tumour tissue, with very low background signal in uMUC-1-negative tumours. However, uMUC1-specific imaging targets the underlying underglycosylated protein, not the glycans directly. The direct imaging of changes in glycosylation might be achieved by tapping into the underlying metabolic processes of the cell. Monosaccharide substrates can be labelled with a non-invasive reporter,

484 | JUNE 2005

| VOLUME 4
2005 Nature Publishing Group

www.nature.com/reviews/drugdisc

REVIEWS

Metabolic labelling
O HO HO HO HN O OH N3

N3

N3

Staudinger ligation
O

N3

H3CO Ph 2P

ManNAz

Cell (altered physiological state)

Cell

Imaging reagent

Figure 4 | Imaging chemically modified cellular glycans in vivo. In a process termed metabolic oligosaccharide engineering, unnatural sugars can be biosynthetically introduced into cellular glycans86,88,89. For example, azide-containing sialicacid residues (SiaNAz) can be metabolically incorporated into membrane glycoconjugates in mice that are treated with the azidosugar precursor N--azidoacetylmannosamine (ManNAz)87. If sialic-acid expression is upregulated in cells with an altered physiological state, such as cancer cells, then these cells should incorporate large amounts of SiaNAz. Subsequent reaction between azide-modified cells and phosphine-conjugated imaging probes (Staudinger ligation; see BOX 2) should allow noninvasive detection of altered tissue. Alternatively, reaction of azide-modified glycans with phosphine-conjugated affinity purification tags should allow enrichment and subsequent identification of glycans on cells with an altered physiological state.

such as a radionuclide (18F or 125I), to follow the metabolic fate of these substrates by radionuclide imaging. Current non-invasive positron-emission tomography imaging technologies for the early diagnosis of cancer rely on the elevated accumulation of the simple sugar 2-fluoro-2-deoxy-glucose (FDG) in tumour tissue relative to healthy tissue83. FDG is an unnatural (18F)-containing glucose analogue that concentrates in malignant tumours owing to their enhanced glycolysis rates. Similarly, the metabolic fates of 14C-radiolabelled monosaccharides have been monitored in animal tumour models, albeit using invasive methods (for example, surgical biopsy)86. We have recently reported the ability to tag cellsurface glycans with probes in vivo87. In a process termed metabolic oligosaccharide engineering, unnatural sugars bearing BIOORTHOGONAL FUNCTIONAL GROUPS can be metabolically introduced into cellular glycans86,88,89. We have adapted this technique for the cell-surface display of glycans bearing azido groups90. The azide is not endogenous to biological systems and is inert to biological components, yet will react with another bioorthogonal functional group, the phosphine, in a selective manner BOX 2. The covalent reaction of azides and phosphines to form a stable adduct is a reaction termed the Staudinger ligation90 BOX 2. This reaction can chemically tag azide-functionalized sugars in living animals87. In these experiments, azide-containing sialic acid residues (SiaNAz) can be metabolically incorporated into membrane glycoconjugates in mice that are treated with the azidosugar precursor N--azidoacetylmannosamine (ManNAz)87. Because cancer cells are known to overexpress sialic acids and are more metabolically active than normal cells, it is possible that these cells would incorporate disproportionately high amounts of SiaNAz into their cell-surface glycans. Subsequent reaction between azide-modified cancer cells and phosphine-conjugated imaging probes would then allow discrimination of cancerous tissue from the

surrounding healthy tissue using non-invasive diagnostic techniques such as magnetic resonance imaging (MRI) (FIG. 4). As a proof-of-principle, Lemieux et al. found that cells expressing high levels of sialic acid could be labelled in cell culture with unnatural sialic acids and preferentially tagged with an MRI-contrast reagent91. Furthermore, changes in glycan expression that accompany the onset or progression of other diseases, such as chronic inflammation, might be identified or tracked non-invasively by chemically tagging glycans in vivo. This chemical tagging could be useful for finding new targets for drug development. More broadly, the azide might serve as an in vivo reporter of glycan expression. In principle, any sugar could metabolically be labelled with an azide if the biosynthetic enzymes are tolerant of azido substrates. In addition to the sialic acid biosynthetic pathway, the N -acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) salvage pathways are tolerant of azido analogues92,93. N--azidoacetylglucosamine and N - -azidoacetylgalactosamine are incorporated into O-GlcNAc-modified proteins and mucin-type O-linked glycoproteins, respectively. We and others are applying this phenomenon to proteomic analysis of glycoprotein subtypes92,93.
Inflammation-associated glycans

In contrast to cancer-associated glycans, where the effects of changes in cellular glycosylation on tumour progression are not fully understood, the functions of glycans found at sites of chronic inflammation are well-defined. Inflammatory diseases, such as arthritis, psoriasis, asthma and diabetes, are characterized by leukocyte homing into the affected tissues. A crucial first step in the normal entry of circulating lymphocytes into peripheral lymph nodes and leukocyte emigration into inflamed tissues is their adhesion to activated endothelial cells lining blood vessel walls94. In addition to their role in cancer metastasis (discussed above), the selectins

NATURE REVIEWS | DRUG DISCOVERY

VOLUME 4 | JUNE 2005 | 485

2005 Nature Publishing Group

REVIEWS
glycosyltransferases105107 and sulphotransferases108 that synthesize sLex or 6-sulpho-sLex) are presently under development. In addition, there is an opportunity for the development of non-invasive diagnostics that might identify sites of chronic inflammation prior to the presentation of disease symptoms. For example, the best diagnostic indicator of type 1 diabetes is the presence of autoantibodies against pancreatic islet cells. By the time these autoantibodies are detectable, a significant amount of autoimmune destruction has already occurred. Weissleder and co-workers have recently reported an improved diagnostic for inflammatory diabetes that targets an earlier event in disease progression leaky vasculature109. Still-earlier detection of inflammatory diabetes might involve imaging the expression of L-selectin ligands, the induction of which presumably precedes inflammatory tissue damage. Sibson et al. reported a strategy for the early detection of inflammation in the brain by targeting the E-selectinsLex interaction110. An MRI-contrast reagent coupled to an sLex mimetic that binds E-selectin revealed the presence of inflammation in the brain that was otherwise invisible by MRI110. Strategies for the early detection of other inflammatory diseases could similarly be improved by imaging the early changes in glycosylation.
Other disease-associated glycans

Table 3 | Induction of sLex and 6-sulpho-sLex in inflammatory diseases


Target organ Synovium Gut Skin Lung Pancreas Other Disease Rheumatoid arthritis Crohns disease; ulcerative colitis Cutaneous sites of inflammation (for example, psoriasis) Bronchiectosis; chronic interstitial pneumonia; asthma Diabetes (NOD mouse model) Transplant rejection References 100,120 121123 100 124 125 126

NOD, non-obese diabetic; sLex, sialyl Lewis x.

(E-, P- and L-selectin) mediate the transient initial interaction that results in rolling of the leukocytes along the endothelial surface9597. The selectins bind sialylated and fucosylated epitopes such as sLex, often in sulphated form, which comprise a terminal component of glycans on most leukocytes, endothelial cells in the lymph node and the endothelium of inflamed tissues98,99. These epitopes have been studied extensively with monoclonal antibodies, such as MECA-79, which binds to a collection of endothelial cell glycoproteins known as peripheral node addressin or PNAd100. MECA-79 binds glycan epitopes in a sulphate-dependent manner and shares similar specificity with L-selectin. MECA-79 and L-selectin ligands (for example, 6-sulpho-sLex) have been shown to be absent from non-inflamed endothelial tissues but are prominently expressed on the endothelium of chronically inflamed tissues TABLE 395,101,102. These glycan epitopes act as endothelial zip codes for the homing of lymphocytes, which constitutively express L-selectin, to inflamed tissues95. Inhibition of leukocyte emigration into inflamed tissue is an attractive target for anti-inflammatory therapy 103 . Like most inhibitors of carbohydratebinding proteins, however, selectin blockers face the challenge of inhibiting multivalent, low-affinity interactions in vivo. Therapeutics that block selectins (for example, selectin inhibitor PSGL-1104) or the biosynthesis of their ligands (for example, inhibitors of the

Although the focus of this article is on changes in glycosylation that occur in diseased tissue within humans, there are also opportunities for targeting the unique glycosylation patterns of pathogenic microbes, such as those that cause malaria or meningitis. Recently, glycan-based vaccine candidates have been developed that activate the hosts immune system against a malarial toxin111 and Haemophilus influenza type b112. Efforts are currently underway to generate glycan-based vaccines that target the viral envelope protein of HIV (gp120)113,114 and other pathogenic microbes, including Salmonella typhi, Candida albicans, Shigella and Mycobacterium tuberculosis115. The unique glycosylation patterns of pathogenic microbes could potentially be targeted for diagnostic purposes as well.

1.

2.

3.

4.

5.

Fabbro, D. & Garcia-Echeverria, C. Targeting protein kinases in cancer therapy. Curr. Opin. Drug Disc. Dev. 5, 701712 (2002). Sliva, D. Signaling pathways responsible for cancer cell invasion as targets for cancer therapy. Curr. Cancer Drug Targ. 4, 327336 (2004). Palladino, M. A., Bahjat, F. R., Theodorakis, E. A. & Moldawer, L. L. Anti-TNF-alpha therapies: The next generation. Nat. Rev. Drug Disc. 2, 736746 (2003). Meezan, E., Wu, H. C., Black, P. H. & Robbins, P. W. Comparative studies on carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. Separation of glycoproteins and glycopeptides by sephadex chromatography. Biochemistry 8, 25182524 (1969). The first demonstration that cancer glycans differ from glycans on healthy cells. Turner, G. A. N-Glycosylation of serum-proteins in disease and its investigation using lectins. Clin. Chim. Acta 208, 149171 (1992).

Axford, J. S. Glycosylation and rheumatic disease. Biochim. Biophys. Acta 1455, 219229 (1999). Mackiewicz, A. & Mackiewicz, K. Glycoforms of serum 1-acid glycoprotein as markers of inflammation and cancer. Glycoconj. J. 12, 241247 (1995). 8. Gabius, H. J. Biological information transfer beyond the genetic code: the sugar code. Natur Wissenschaften 87, 108121 (2000). 9. Saussez, S. et al. Quantitative glycohistochemistry defines new prognostic markers for cancers of the oral cavity. Cancer 82, 252260 (1998). 10. Shriver, Z., Raguram, S. & Sasisekharan, R. Glycomics: a pathway to a class of new and improved therapeutics. Nat. Rev. Drug Disc. 3, 863873 (2004). 11. Pancino, G. et al. Purification and characterization of a breast-cancer-associated glycoprotein not expressed in normal breast and identified by monoclonal antibody83d4. Brit. J. Cancer 63, 390398 (1991). 12. Matsushita, Y., Cleary, K. R., Ota, D. M., Hoff, S. D. & Irimura, T. Sialyl-dimeric Lewis-X antigen expressed on 7.

6.

mucin-like glycoproteins in colorectal-cancer metastases. Lab. Invest. 63, 780791 (1990). 13. Dennis, J. W., Laferte, S., Waghorne, C., Breitman, M. L. & Kerbel, R. S. 1-6 branching of asn-linked oligosaccharides is directly associated with metastasis. Science 236, 582585 (1987). 14. Kim, Y. J. & Varki, A. Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconj. J. 14, 569576 (1997). 15. Sell, S. Cancer-associated carbohydrates identified by monoclonal antibodies. Human Path. 21, 10031019 (1990). 16. Hakomori, S. & Zhang, Y. Glycosphingolipid antigens and cancer therapy. Chem. Biol. 4, 97-104 (1997). 17. Taylorpapadimitriou, J. & Epenetos, A. A. Exploiting altered glycosylation patterns in cancer- progress and challenges in diagnosis and therapy. Trends Biotech. 12, 227233 (1994). 18. Gabius, H. J. Tumor lectinology- at the intersection of carbohydrate chemistry, biochemistry, cell biology, and oncology. Angew. Chem. 27, 12671276 (1988).

486 | JUNE 2005

| VOLUME 4
2005 Nature Publishing Group

www.nature.com/reviews/drugdisc

REVIEWS

19. Orntoft, T. F. & Vestergaard, E. M. Clinical aspects of altered glycosylation of glycoproteins in cancer. Electrophoresis 20, 362371 (1999). 20. Hollingsworth, M. A. & Swanson, B. J. Mucins in cancer: Protection and control of the cell surface. Nat. Rev. Cancer 4, 45-60 (2004). 21. Hakomori, S. Traveling for the glycosphingolipid path. Glycoconj. J. 17, 627647 (2000). 22. Zhang, S. L. et al. Selection of tumor antigens as targets for immune attack using immunohistochemistry. 1. Focus on gangliosides. Int. J. Cancer 73, 42-49 (1997). 23. Zhang, S. L. et al. Selection of tumor antigens as targets for immune attack using immunohistochemistry. 2. Blood grouprelated antigens. Int. J. Cancer 73, 50-56 (1997). 24. Fukuda, M. Possible roles of tumor-associated carbohydrate antigens. Cancer Res. 56, 223744 (1996). 25. Jurianz, K. et al. Complement resistance of tumor cells: Basal and induced mechanisms. Mol. Immunol. 36, 929 939 (1999). 26. Speiser, D. E. et al. Self antigens expressed by solid tumors do not efficiently stimulate naive or activated T cells: Implications for immunotherapy. J. Exp. Med. 186, 645653 (1997). 27. Danishefsky, S. J. & Allen, J. R. From the laboratory to the clinic: A retrospective on fully synthetic carbohydratebased anticancer vaccines. Angew. Chem. 39, 836863 (2000). An excellent review of synthetic carbohydrate-based anticancer vaccines. 28. Livingston, P. O. Approaches to augmenting the immunogenicity of melanoma gangliosides- from whole melanoma-cells to ganglioside-KLH conjugate vaccines. Immunol. Rev. 145, 147166 (1995). 29. Ragupathi, G. et al. On the power of chemical synthesis: Immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines. Proc. Natl Acad. Sci. U. S. A. 99, 1369913704 (2002). 30. Gilewski, T. et al. Immunization of metastatic breast cancer patients with a fully synthetic globe H conjugate: A phase I trial. Proc. Natl Acad. Sci. U. S. A. 98, 32703275 (2001). 31. Slovin, S. F. et al. Carbohydrate vaccines in cancer: Immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man. Proc. Natl Acad. Sci. U. S. A. 96, 57105715 (1999). 32. Chapman, P. B. et al. A phase II trial comparing five dose levels of BEC2 anti-idiotypic monoclonal antibody vaccine that mimics GD3 ganglioside. Vaccine 22, 29042909 (2004). 33. Krug, L. M. et al. Vaccination of small cell lung cancer patients with polysialic acid or N-propionylated polysialic acid conjugated to keyhole limpet hemocyanin. Clin. Cancer Res. 10, 916923 (2004). The first demonstration in humans that vaccines containing unnatural sugars mount a more robust immune response against cancer glycans than their natural counterparts. 34. Ragupathi, G. et al. Consistent antibody response against ganglioside GD2 induced in patients with melanoma by a GD2 lactone-keyhole limpet hemocyanin conjugate vaccine plus immunological adjuvant QS-21. Clin. Cancer Res. 9, 52145220 (2003). 35. Dove, A. The bittersweet promise of glycobiology. Nat. Biotech. 19, 913917 (2001). 36. Holmberg, L. & Sandmaier, B. Vaccination with Theratope (STn-KLH) as treatment for breast cancer. Expert Rev. Vaccines 3, 655663 (2004). 37. Livingston, P. O. The unfulfilled promise of melanoma vaccines. Clin. Cancer Res. 7, 18371838 (2001). 38. Slovin, S. F. et al. Fully synthetic carbohydrate-based vaccines in biochemically relapsed prostate cancer: Clinical trial results with -N-acetylgalactosamine-Oserine/threonine conjugate vaccine. J. Clin. Oncol. 21, 42924298 (2003). 39. Liu, T. M., Guo, Z. W., Yang, Q. L., Sad, S. & Jennings, H. J. Biochemical engineering of surface 2,8 polysialic acid for immunotargeting tumor cells. J. Biol. Chem. 275, 3283232836 (2000). The first report on the use of unnatural sialic acid biosynthesis for tumour immunotherapy in animals. 40. Chefalo, P., Pan, Y. B., Nagy, N., Harding, C. & Guo, Z. W. Preparation and immunological studies of protein conjugates of N-acylneuraminic acids. Glycoconj. J. 20, 407414 (2003). 41. Pan, Y. B., Chefalo, P., Nagy, N., Harding, C. & Guo, Z. W. Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines. J. Med. Chem. 48, 875883 (2005). 42. Zou, W. et al. Bioengineering of surface GD3 ganglioside for immunotargeting human melanoma cells. J. Biol. Chem. 279, 2539025399 (2004).

43. Lemieux, G. A. & Bertozzi, C. R. Modulating cell surface immunoreactivity by metabolic induction of unnatural carbohydrate antigens. Chem. Biol. 8, 265275 (2001). 44. Galili, U. Autologous tumor vaccines processed to express -gal epitopes: a practical approach to immunotherapy in cancer. Cancer Immunol. Immunother. 53, 935945 (2004). 45. Galili, U., Chen, Z. C. & DeGeest, K. Expression of -gal epitopes on ovarian carcinoma membranes to be used as a novel autologous tumor vaccine. Gynecol. Oncol. 90, 100108 (2003). 46. Deriy, L., Chen, Z. C., Gao, G. P. & Galili, U. Expression of -gal epitopes on HeLa cells transduced with adenovirus containing 1,3galactosyltransferase cDNA. Glycobiology 12, 135144 (2002). 47. Deriy, L., Ogawa, H., Gao, G. P. & Galili, U. In vivo targeting of vaccinating tumor cells to antigenpresenting cells by a gene therapy method with adenovirus containing the 1,3 galactosyltransferase gene. Cancer Gene Ther. (in press). 48. Burchell, J. M., Mungul, A. & Taylor-Papadimitriou, J. O-linked glycosylation in the mammary gland: Changes that occur during malignancy. J. Mamm. Gland Biol. Neopl. 6, 355364 (2001). 49. Wong, N. K. et al. Characterization of the oligosaccharides associated with the human ovarian tumor marker CA125. J. Biol. Chem. 278, 2861928634 (2003). 50. Basu, P. S., Majhi, R. & Batabyal, S. K. Lectin and serumPSA interaction as a screeninng test for prostate cancer. Clin. Biochem. 36, 373376 (2003). 51. Peracaula, R. et al. Altered glycosylation pattern allows the distinction between prostate-specifc antigen (PSA) from normal and tumor origins. Glycobiology 13, 457470 (2003). 52. MacDonald, J. S. Carcinoembryonic antigen screening: Pros and cons. Sem. Oncol. 26, 556560 (1999). 53. Lloyd, K. O., Burchell, J., Kudryashov, V., Yin, B. W. & Taylor-Papadimitriou, J. Comparison of O-linked carbohydrate chains in MUC-1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines. Demonstration of simpler and fewer glycan chains in tumor cells. J. Biol. Chem. 271, 3332534 (1996). 54. Dalziel, M. et al. The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1. J. Biol. Chem. 276, 1100711015 (2001). 55. Ramanathan, R. K. et al. Phase I study of a MUC1 vaccine composed of different doses of MUC1 peptide with SB-AS2 adjuvant in resected and locally advance pancreatic cancer. Cancer Immunol. Immunother. 54, 254264 (2005). 56. Karanikas, V. et al. Antibody and T cell responses of patients with adenocarcinoma immunized with mannanMUC1 fusion protein. J. Clin. Invest. 100, 27832792 (1997). 57. Xing, P. X. et al. Phase I study of synthetic MUC1 peptides in cancer. Int. J. Oncol. 6, 12831289 (1995). 58. Renkonen, J., Paavonen, T. & Renkonen, R. Endothelial and epithelial expression of sialyl Lewis(x) and sialyl Lewis(a) in lesions of breast carcinoma. Int. J. Cancer 74, 296300 (1997). 59. Miyake, M., Taki, T., Hitomi, S. & Hakomori, S. Correlation of expression of H/Le(Y)/Le(B) antigens with survival in patients with carcinoma of the lung. New Eng. J. Med. 327, 14-18 (1992). 60. Nakamori, S. et al. Increased expression of sialyl Lewis(X) antigen correlates with poor survival in patients with colorectal-carcinoma- clinicopathological and immunohistochemical study. Cancer Res. 53, 36323637 (1993). 61. Shimodaira, K. et al. Carcinoma-associated expression of core 2 1,6-N-acetylglucosaminyltransferase gene in human colorectal cancer: Role of O-glycans in tumor progression. Cancer Res. 57, 52015206 (1997). 62. Gorelik, E., Galili, U. & Raz, A. On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis. Cancer Metast. Rev. 20, 245277 (2001). 63. Guo, H. B., Lee, I., Kamar, M. & Pierce, M. N-Acetylglucos aminyltransferase V expression levels regulate cadherinassociated homotypic cell-cell adhesion and intracellular signaling pathways. J. Biol. Chem. 278, 5241252424 (2004). 64. Demetriou, M., Nabi, I. R., Coppolino, M., Dedhar, S. & Dennis, J. W. Reduced contact-inhibition and substratum adhesion in epithelial-cells expressing GlcNAc-transferaseV. J. Cell Biol. 130, 383392 (1995). 65. Stanley, P. Selection of lectin-resistant mutants of animalcells. Meth. Enz. 96, 157184 (1983). 66. Tao, T. W. & Burger, M. M. Non-metastasizing variants selected from metastasizing melanoma cells. Nature 270, 437438 (1977).

67. Tokuyama, S. et al. Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA. Int. J. Cancer 73, 410415 (1997). 68. Granovsky, M. et al. Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nature Med. 6, 306 312 (2000). This paper describes reduced mammary tumour growth and metastasis in Mgat5/ mice than in transgenic littermates expressing Mgat5, supporting the role of the branching 1,6GlcNAc residue in cancer progression. 69. Tang, D. G. & Honn, K. V. Adhesion molecules and tumormetastasis- an update. Inv. Metast. 14, 109122 (1994). 70. McEver, R. P. Selectin-carbohydrate interactions during inflammation and metastasis. Glycoconj. J. 14, 585591 (1997). 71. Foster, M. M., Brown, J. R., Wang, L. C. & Esko, J. D. A disaccharide precursor of sialyl Lewis X inhibits metastatic potential of tumor cells. Cancer Res. 63, 27752781 (2003). A description of a small molecule glycosylation inhibitor capable of decreasing metastasis in a murine tumour model. 72. Sarkar, A. K., Rostand, K. S., Jain, R. K., Matta, K. L. & Esko, J. D. Fucosylation of disaccharide precursors of sialyl Lewis(X) inhibit selectin-mediated cell adhesion. J. Biol. Chem. 272, 2560825616 (1997). 73. Brown, J. R., Fuster, M. M., Whisenant, T. & Esko, J. D. Expression patterns of 2,3-sialyltransferases and 1,3fucosyltransferases determine the mode of sialyl Lewis X inhibition by disaccharide decoys. J. Biol. Chem. 278, 2335223359 (2003). 74. Alper, J. Glycobiology- Turning sweet on cancer. Science 301, 159160 (2003). 75. Borsig, L. et al. Heparin and cancer revisited: Mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis. Proc. Natl Acad. Sci. U. S. A. 98, 33523357 (2001). 76. Borsig, L., Kim, Y. J., Varki, N. & Varki, A. The role of P-selectin and carcinoma mucins in tumor growth and metastasis. Glycobiology 8, 11431143 (1998). This report showed that heparin inhibits metastasis by interrupting glycan-mediated platelettumour cell adhesion. 77. Kim, Y. J., Borsig, L., Varki, N. M. & Varki, A. P-selectin deficiency attenuates tumor growth and metastasis. Proc. Natl Acad. Sci. U. S. A. 95, 93259330 (1998). The authors demonstrate that P-selectin-deficient mice showed significantly slower growth of subcutaneously implanted human colon carcinoma cells and generated fewer lung metastases from intravenously injected cells than P-selectinexpressing littermates. 78. Shirota, K., Kato, Y., Irimura, T., Konda, H. & Sugiyama, Y. Anti-metastatic effect of the sialyl lewis-X analog GSC-150 on the human colon carcinoma derived cell line KM12-HX in the mouse. Biol. Pharm. Bull. 24, 316319 (2001). 79. Zacharski, L. R. & Ornstein, D. L. Heparin and cancer. Thromb. Haemost. 80, 10-23 (1998). 80. Nitti, D. et al. Final results of a phase III clinical trial on adjuvant intraportal infusion with heparin and 5-fluorouracil (5-FU) in resectable colon cancer. European Journal of Cancer 33, 12091215 (1997). 81. Weissleder, R. Scaling down imaging: molecular mapping of cancer in mice. Nat. Rev. Cancer 2, 11-8 (2002). 82. Weissleder, R. & Ntziachristos, V. Shedding light onto live molecular targets. Nat. Med. 9, 1238 (2003). 83. Gambhir, S. S. Molecular imaging of cancer with positron emission tomography. Nat. Rev. Cancer 2, 68393 (2002). 84. Sivolapenko, G. et al. Breast cancer imaging with radiolabelled peptide from complementarydetermining region of antitumor immunity. Lancet 346 (1995). 85. Moore, A., Medarova, Z., Potthast, A. & Dai, G. P. In vivo targeting of underglycosylated MUC-1 tumor antigen using a multimodal imaging probe. Cancer Research 64, 18211827 (2004). This report describes noninvasive imaging of breast cancer in a murine tumour model using a peptide that recognizes uMUC1. 86. Kayser, H. et al. Biosynthesis of a nonphysiological sialicacid in different rat organs, using N-propanoyl-Dhexosamines as precursors. J. Biol. Chem. 267, 16934 16938 (1992). 87. Prescher, J. P., Dube, D. H. & Bertozzi, C. R. Chemical remodeling of cell surfaces in living animals. Nature 430, 873877 (2004). The first demonstration that the Staudinger ligation can tag azide-containing glycans in vivo. 88. Dube, D. H. & Bertozzi, C. R. Metabolic oligosaccharide engineering as a tool for glycobiology. Curr. Opin. Chem. Biol. 7, 616625 (2003).

NATURE REVIEWS | DRUG DISCOVERY

VOLUME 4 | JUNE 2005 | 487

2005 Nature Publishing Group

REVIEWS

89. Keppler, O. T., Horstkorte, R., Pawlita, M., Schmidts, C. & Reutter, W. Biochemical engineering of the N-acyl side chain of sialic acid: biological implications. Glycobiology 11, 11R18R (2001). 90. Saxon, E. & Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 287, 20072010 (2000). 91. Lemieux, G. A., Yarema, K. J., Jacobs, C. L. & Bertozzi, C. R. Exploiting differences in sialoside expression for selective targeting of MRI contrast reagents. J. Am. Chem. Soc. 121, 42784279 (1999). 92. Vocadlo, D. J., Hang, H. C., Kim, E. J., Hanover, J. A. & Bertozzi, C. R. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc. Natl Acad. Sci. U. S. A. 100, 91169121 (2003). 93. Hang, H. C., Yu, C., Kato, D. L. & Bertozzi, C. R. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation. Proc. Natl Acad. Sci. U. S. A. 100, 1484614851 (2003). 94. Kansas, G. S. Selectins and their ligands: Current concepts and controversies. Blood 88, 32593287 (1996). 95. Renkonen, J., Tynninen, O., Hayry, P., Paavonen, T. & Renkonen, R. Glycosylation might provide endothelial zip codes for organ-specific leukocyte traffic into inflammatory sites. Am. J. Pathol. 161, 543550 (2002). 96. Lowe, J. B. Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr. Opin. Cell Biol. 15, 531 538 (2003). 97. Ley, K. The role of selectins in inflammation and disease. Trends Mol. Med. 9, 263268 (2003). 98. Crocker, P. R. & Feizi, T. Carbohydrate recognition systems: Functional triads in cell-cell interactions. Curr. Opin. Struct. Biol. 6, 679691 (1996). 99. Varki, A. Selectin ligands. Proc. Natl Acad. Sci. U. S. A. 91, 73907397 (1994). 100. Michie, S. A., Streeter, P. R., Bolt, P. A., Butcher, E. C. & Picker, L. J. The human peripheral lymph-node vascular addressin- an inducible endothelial antigen involved in lymphocyte homing. Am. J. Pathol. 143, 16881698 (1993). 101. Rosen, S. D. Endothelial ligands for L-selectin: From lymphocyte recirculation to allograft rejection. Am. J. Pathol. 155, 10131020 (1999). 102. Kannagi, R. Regulatory roles of carbohydrate ligands for selectins in the homing of lymphocytes. Curr. Opin. Struct. Biol. 12, 599608 (2002). 103. Zollner, T. M. & Asadullah, K. Selectin and selectin ligand binding: a bittersweet attraction. J. Clin. Invest. 112, 980 983 (2003). 104. Khor, S. P., McCarthy, K., Dupont, M., Murray, K. & Timony, G. Pharmacokinetics, pharmacodynamics, allometry, and dose selection of rPSGL-Ig for phase I trial. J. Pharm. Exper. Ther. 293, 618624 (2000).

105. Lee, L. V. et al. A potent and highly selective inhibitor of human 1,3-fucosyltransferase via click chemistry. J. Am. Chem. Soc. 125, 95889589 (2003). 106. Lin, C. H. et al. Enzymatic synthesis of a sialyl Lewis X dimer from egg yolk as an inhibitor of E-selectin. Bioorg. Med. Chem. 3, 16251630 (1995). 107. Dimitroff, C. J., Kupper, T. S. & Sackstein, R. Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen. J. Clin. Invest. 112, 10081018 (2003). 108. Armstrong, J. I. et al. A library approach to the generation of bisubstrate analoge sulfotransferse inhibitors. Org. Lett. 23, 26572660 (2001). 109. Denis, M. C., Mahmood, U., Benoist, C., Mathis, D. & Weissleder, R. Imaging inflammation of the pancreatic islets in type 1 diabetes. Proc. Natl Acad. Sci. U. S. A. 101, 1263412639 (2004). 110. Sibson, N. R. et al. MRI detection of early endothelial activation in brain inflammation. Mag. Reson. Med. 51, 248252 (2004). 111. Schofield, L., Hewitt, M. C., Evans, K., Siomos, M. A. & Seeberger, P. H. Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria. Nature 418, 785789 (2002). 112. Verez-Bencomo, V. et al. A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b. Science 305, 522525 (2004). 113. Mandal, M., Dudkin, V. Y., Geng, X. D. & Danishefsky, S. In pursuit of carbohydrate-based HIV vaccines, Part 1: The total synthesis of hybrid-type gp120 fragments. Angew. Chem. 43, 25572561 (2004). 114. Geng, X. D., Dudkin, V. Y., Mandal, M. & Danishefsky, S. J. In pursuit of carbohydrate-based HIV vaccines, Part 2: The total synthesis of high-mannose-type gp120 fragmentsevaluation of strategies directed to maximal convergence. Angew. Chem. 43, 25622565 (2004). 115. Borman, S. Carbohydrate vaccines: novel chemical and enzymatic oligosaccharide synthesis techniques could lead to a new generation of carbohydratebased vaccine agents. Chem. Eng. News 82, 31-35 (2004). 116. Knutson, K. L. GMK (Progenics Pharmaceuticals). Curr. Opin. Invest. Drugs 3, 159164 (2002). 117. Ragupathi, G. et al. A fully synthetic globo H carbohydrate vaccine induces a focused humoral response in prostate cancer patients: A proof of principle. Angew. Chem. 38, 563566 (1999). 118. Krug, L. M. et al. Vaccination of patients with small-cell lung cancer with synthetic fucosyl GM-1 conjugated to keyhole limpet hemocyanin. Clin. Cancer Res. 10, 6094 6100 (2004). 119. Sabbatini, P. J. et al. Immunization of ovarian cancer patients with a synthetic Lewis(Y)protein conjugate vaccine: A phase 1 trial. Int. J. Cancer 87, 79-85 (2000).

120. Vandintherjanssen, A., Pals, S. T., Scheper, R., Breedveld, F. & Meijer, C. Dendritic cells and high endothelial venules in the rheumatoid synovialmembrane. J. Rhematol. 17, 11-17 (1990). 121. Salmi, M., Granfors, K., Macdermott, R. & Jalkanen, S. Aberrant binding of lamina propria lymphocytes to vascular endothelium in inflammatory bowel diseases. Gastroenterology 106, 596605 (1994). 122. Salmi, M. & Jalkanen, S. Regulation of L-selectin expression on cultured bone-marrow leukocytes and their precursors. Eur. J. Immunol. 22, 835843 (1992). 123. Duijvestijn, A. M., Kerkhove, M., Bargatze, R. F. & Butcher, E. C. Lymphoid tissue-specific and inflammation-specific endothelial-cell differentiation defined by monoclonalantibodies. J. Immunol. 138, 713719 (1987). 124. Toppila, S., Paavonen, T., Laitinen, A., Laitinen, L. A. & Renkonen, R. Endothelial sulfated sialyl Lewis X glycans, putative L-selectin ligands, are preferentially expressed in bronchial asthma but not in other chronic inflammatory lung diseases. Am. J. Respir. Cell Mol. Biol. 23, 492498 (2000). 125. Hanninen, A. et al. Vascular addressins are induced on islet vessels during insulitis in nonobese diabetic mice and are involved in lymphoid-cell binding to islet endothelium. J. Clin. Invest. 92, 25092515 (1993). 126. Turunen, J. P. et al. De-novo expression of endothelial sialyl Lewis(a) and sialyl Lewis(X) during cardiac transplant rejection- superior capacity of a tetravalent sialyl Lewis(X) oligosaccharide in inhibiting L-selectindependent lymphocyte adhesion. J. Exp. Med. 182, 11331141 (1995). 127 Slovin, S. et al. Multivalency in a phase II prostate cancer (PC) vaccine trial: Are more antigens better? Proc. Am. Soc. Clin. Oncol. 22, 167 (2003).

Acknowledgements
We thank J. Prescher, M. Paulick, I. Miller and S. Laughlin for critical reading of the manuscript.

Competing interests statement


The authors declare no competing financial interests.

Online links
DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene E-selectin | GlcNAc-TV | L-selectin | MUC1 | MUC16 | prostatespecific antigen | P-selectin | ST3Gal1 OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM Arthritis | asthma | diabetes | psoriasis Access to this interactive links box is free online.

488 | JUNE 2005

| VOLUME 4
2005 Nature Publishing Group

www.nature.com/reviews/drugdisc

Você também pode gostar