Fabio Dall’Olio · 2012. 10. 26. · From: Trinchera et al. 2011 By immunoblot analysis, only a...

BIOSYNTHESIS OF CANCER-RELATED

CARBOHYDRATE ANTIGENS

Fabio Dall’OlioDepartment of Experimental Pathology

University of Bologna, Italy

)

TOPICS OF THE LECTURE

1. Structure and function of some representative cancer-associated

carbohydrate antigens

β1,6-branchingSialyl-Lewis antigens

Sia6LacNAc

2. Mechanisms leading to the expression of cancer-associated carbohydrate

antigens

Overexpression of glycosyltransferases

Altered glycosidase expression

Masking of sugar structures by substituent groups

Competition between normal and cancer-associated carbohydrate structures

3. Glycosylation and cell signalling: a bi-directional talk

)

1. SOME CANCER-ASSOCIATED


ββββ1,6-branchingSialyl-Lewis antigens

Sia6LacNAc

)

Siaαααα2,3(6)Galββββ1,4GlcNAcββββ1,2Man

Manββββ1,4GlcNAcββββ1,4GlcNAc-Asn


Siaαααα2,3Galββββ1,4GlcNAcββββ1,3(Galββββ1,4GlcNAc)nββββ1,3Galββββ1,4GlcNAcββββ1,6

Fucαααα1,3


Manββββ1,4GlcNAcββββ1,4GlcNAc-Asn


GnT5

GnT3

Bisecting GlcNAc

ββββ1,6-branching


GlcNAcββββ1,4Manββββ1,4GlcNAcββββ1,4GlcNAc-Asn


ββββ1,6 BRANCHING AND BISECTING GlcNAc ARE

ALTERNATIVE STRUCTURES

An N-linked chain can be transformed in a β1,6-branched structure (upper) by the action of GnT5.

The β1,6-linked branch is frequently elongated by polylactosaminic chains, which are often

terminated by complex structures, such as the sialyl Lex antigen. The action of GnT3 leads to the

addition of a bisecting GlcNAc, which inhibits the formation of the β1,6-branched structure.

From: Dall’Olio et al. Carbohydr. Chem. 2012 37 21-56

ββββ1,6-BRANCHING AND METASTASIS

1. The ββββ1,6-branching, as detected with

leukoagglutinin (L-PHA), is associated with

metastasis (Dennis et al. 1987)

2. GnT5 is controlled by ras and other oncogenes (Lu

et al. 1993).

3. GnT5 null mice in a PyMT background display

strong inhibition of tumor growth and metastasis

(Granowsky et al. 2000)

4. ββββ1,6-branched glycans on growth factor receptors

enhance their signalling, through interaction with

galectin-3 (Lau et al. 2007).

5. ββββ1,6-branched glycans stabilize matriptase, an

activator of hepatocyte growth factor (Ihara et al.

2004)

)

SIALYL LEWIS ANTIGENSThe basic units of

type 1 or type 2

chains are obtained

by the addition on

GlcNAc of a β1,3-

linked or a β1,4-linked

galactose,

respectively. The

α2,3-sialylation of

type 1 or 2 chains,

followed by the

addition of α1,4- or

α1,3-linked fucose,

respectively, leads to

the biosynthesis of

the sialyl Lea (sLea)

and sialyl Lex (sLex)

antigens.

)

ROLE OF SELECTIN INTERACTIONS

DURING CELL EXTRAVASATION

Selectin ligands sLea/sLex on circulating cells interact with E and P-

selectins expressed by activated endothelia. These interactions

mediate the tethering and rolling of leukocytes or cancer cells. The

firm adhesion, which precedes extravasation is mediated by

integrins.

)

SIALYL LEWIS ANTIGENS AND CANCER

1.The up-regulation of sialyl Lewis

antigens (sLe) is frequently observed in

cancer (Izawa et al. 2000)

2.sLea (CA19.9) is a widely used tumor

marker

3.In many systems sLe antigens are

associated with metastasis

4.sLe antigens are selectin-ligands

)

From: Izawa et al. 2000

SIALYL LEWIS-X ANTIGEN IS

OVEREXPRESSED IN COLON CANCER

Staining with the anti sLex

antibody CSLEX reveals

that sLex is expressed only

by cancer tissues.

Patient

normaltumor

1 2 3 4 5 6 7 8 9 1011121314151617

5

2.5

0

sL

ex

(a

rbit

rary

un

its

)

From: Trinchera et al. 2011

By immunoblot analysis,

only a few colon cancer

tissues express high

levels of sLex

)

Manββββ1,4GlcNAcββββ1,4GlcNAc---Asn

Galββββ1,4GlcNAcββββ1,

Galββββ1,4GlcNAcββββ1,

Galββββ1,4GlcNAcββββ1,2

6

3

Manαααα1

Manαααα1

6

3

Siaαααα2,6

Siaαααα2,6

Siaαααα2,6

αααα2,6-SIALYLATED LACTOSAMINE (Sia6LacNAc)

Sialic acid can be linked to lactosaminic chains either

through a αααα2,3- or a αααα2,6-linkage.

ST6Gal.1 mediates this reaction on glycoproteins

Sambucus nigra agglutinin (SNA) recognizes

Sia6LacNAc

)

Sia6LacNAc AND CANCER

1.The up-regulation of ST6Gal.1 and/or

Sia6LacNAc is frequently observed in

cancers (Dall’Olio 2000a, Dall’Olio et al.

2001)

2.The ββββ1 subunit of integrins is a substrate

of ST6Gal.1 (Seales et al. 2005)

3.High ST6Gal.1 induces radiation

resistance (Lee et al. 2008)

4.Breast cancers developed by ST6Gal.1

null mice in a PyMT background are more

differentiated (Hedlund et al. 2008)

)

ST6Gal.1 AND αααα2,6 SIALYLATED SUGAR

CHAINS INCREASE IN COLORECTAL CANCER

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Normale

Tumorale

ST6Gal.I activity increases in nearly

100% of the colorectal cancer cases

although at a variable level among

patients (Dall’Olio et al. 1989, 2000b)

The increase of Sia6LacNAc

expression, detected with SNA,

marks the transition between normal

and cancer tissue (Dall’Olio and

Trerè 1993)

ST6Gal.I IS UNDER THE CONTROL

OF THE RAS ONCOGENE

Northern blot analysis

of mock-transfected

and K-ras transfected

NIH3T3 cells with

probes for different

sialyltransferases.

Only ST6Gal.I shows

ras-dependent

modulation

SNA FACS

analysis of mock-

transfected and K-

ras transfected

NIH3T3 cells. Ras

expression

induces αααα2,6-

sialylation of cell

membrane.

From: Dalziel et al. 2004

)

Mock ST6Gal.1+

Day 0

Day 6

PHENOTYPIC CHANGES INDUCED BY ST6Gal.1

EXPRESSION IN SW948 COLON CANCER CELLS

From: Chiricolo et al. 2006

0

0,5

1

1,5

3,5 7 14

Fibronectin (µg/well)

A6

50

0

0,5

1

1,5

2

2,5

14 21 42

Collagen IV (µg/well)

A6

50

**

**

*

Fibronectin

Collagen IV

A B C

A: adhesion to ECM components (grey=mock;

black=ST6Gal.1+)

B: scratch wound assay

C: SNA reactivity (left) and morphology (right) of

ST6Gal.1+ cells.

)

2. MECHANISMS DRIVING THE

EXPRESSION OF CANCER-RELATED


Overexpression of glycosyltransferases

Altered glycosidase expression

Masking by substituent groups

Competition between glycosyltransferases

)

THE OVEREXPRESSION OF CARBOHYDRATE

STRUCTURES IS USUALLY MULTIFACTORIAL

Structure

overexpression

Cognate glycosyltransferase↑

Competing glycosyltransferases ↓

Glycosidases ↓Masking by substituents ↓e.g.: Neu sialidases

e.g.: ST6Gal.1

GnT5

e.g.: ββββ4GalNAcT2/sLex

GnT3/ββββ1,6-branching

e.g.: O-acetylation of sialic acids

)

INCREASE OF COGNATE GLYCOSYLTRANSFERASE:

ST6Gal.1 AND SNA REACTIVITY

From: Dall’Olio et al. Glycobiology 2004

The SNA reactivity and the

ST6Gal.1 activity of 21 pairs

of cancer and non-cancer

liver tissues were

determined and plotted.

The relationship between

SNA and ST6Gal.1 is non

linear, indicating that

factors other than ST6Gal.1

activity affect the

biosynthesis of Sia6LacNAc

)

MASKING BY SUBSTITUENTS: O-

ACETYLATION OF SIALIC ACIDS

Untreated

N T N T N T N T N T N T N T N T N T N T

Patient number

2.5 µµµµg

1.2 µµµµg

0.6 µµµµg

1 2 3 4 5 6 7 8 9 10

Alkali-treated

anti sLex

O-acetyl groups on sialic acids can hinder the recognition of sialylated

sugar antigens by antibodies (Ogata et al. 1995). Removal of O- acetyl

groups by alkali enhances the detection of sLex in normal colon

From: Malagolini et al. Glycobiology 2007

2.5 µµµµg

1.2 µµµµg

0.6 µµµµg

)

DECREASED GLYCOSIDASE

EXPRESSION: SIALIDASES

1.Neu sialidases are a group of 4 enzymes

with different intracellular localization

2.Downregulation of lysosomal Neu1 in

cancer promotes anchorage-independent

growth and metastatic ability

3.Over-expression of cytosolic Neu2 leads

to reduced invasion of cancer cells

(Miyagi et al. 2008)

)

COMPETITION BETWEEN THE sLex AND

Sda ANTIGENS IN COLON CANCER

normal tumor5

2.5

sLex

P<0.0001

Attiv

ità F

ucT

-VI

0

5

10

3 6sLex

tumor

normal

sLex0

4

8

0.3 0.6

NS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

12

6

0

FucT

-VI activ

ity

Patient

A

B

D

C

Attiv

ità F

ucT

-VI

A: sLex is highly

expressed by some

cancer tissues and

very little by normal

tissue.

B: Its biosynthetic

enzyme (FucT-VI) is

high in both normal

and cancer.

sLex correlates with

FucT-VI in cancer (C)

but not in normal

tissue (D).

From: Trinchera et al. 2011

)

ENZIMATIC BASIS OF REDUCED sLex

BIOSYNTHESIS IN NORMAL COLON

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

Patient

1

2

Attiv

itàββ ββ4GalN

AcT

-II

normal tumor

ββββ4GalNAcT-II is down-

regulated in colon cancer

tissuesFucT

-VI/ββ ββ4GalN

AcT

-II

sLex

P<0.0001

In normal colon sLex

correlates with the FucT-

VI/ ββββ4GalNAcT-II ratio

❠❠

Sda AND sLex ANTIGENS

Neu5Acαααα2,3Galββββ1,4GlcNAc-R

“Good” Sda

“Bad” sLex

GalNAcββββ1,4


Fucαααα1,3


❠❠

Expression of ββββ4GalNAcT-II

results in sLex inhibition in dot

blot analysis (A), FACS (B) and

fluorescence microscopy (C).

ββββ4GalNAcT-II INHIBITS sLex BIOSYNTHESIS IN

LS174T COLON CANCER CELLS

Mock

25 12.5 6 3 1.5 0.7

µµµµg homogenate

Anti sLex

Mock ββββ4GalNAcT-II+A

nti S

da

An

ti sL

ex

ββββ4GalNAcT-II+

mock

ββββ4GalNAcT-II+

Malagolini et al. 2007

A

B

C

)

Sda

sLex

Normal Tumor

FucT-VI

ββββ4GalNAcT-II

ββββ4GalNAcT-II

FucT-VI

Reduced ββββ4GalNAcT-II expression in cancer tissue results in sLex

overexpression even in the presence of constant FucT-VI activity

BALANCE BETWEEN Sda AND

sLex ANTIGENS IN COLON

ʐ❠

3. GLYCOSYLATION AND CELL

SIGNALLING: A BI-DIRECTIONAL TALK

The cancer-related carbohydrate

changes can be at the same time

the cause and the effect of the

malignant phenotype

❠❠

DYNAMIC OF CANCER-ASSOCIATED

GLYCOSYLATION CHANGES

Cell

transformation

Glycosylation

changes

Inside out

Outside in

Altered

phenotype

Cancer

markers

)

Sia6LacNAc ββββ1,6-branching

Integrins

Growth Factor Receptors

Oncogene signalling

EXAMPLE OF BI-DIRECTIONAL SIGNALLING

Outside-in signalling. Sugar

structures, such as Sia6

LacNAc and ββββ1,6-branching,

decorate cell surface molecules,

including integrins and growth

factor receptors. This results in

a potentiation of their signalling,

eventually leading to oncogene

activation.

Inside-out signalling.

Oncogenes like ras, directly up-

regulate the expression of the

enzymes (ST6Gal.1 and GnT5)

which synthesize Sia6LacNAc

and ββββ1,6-branching.

This bidirectional signalling

might create a self-fuelling loop.

❠❠

ESSENTIAL BIBLIOGRAPHY

Chiricolo et al. Glycobiology 16, 146-154 (2006)

Dall’Olio et al. Int. J. Cancer 44, 434-439 (1989)

Dall’Olio and Trerè Eur. J. Histochem. 37, 257-265 (1993)

Dall’Olio Glycoconj. J. 17,669-676 (2000)

Dall’Olio et al. Int. J. Cancer 88, 58-65 (2000)

Dall’Olio and Chiricolo Glycoconj. J. 18, 841-850 (2001)

Dall’Olio et al. Glycobiology 14, 39-49 (2004)

Dall’Olio F. et al. Front. Biosci. 17, 670-699 (2012)

Dall’Olio et al. Carbohydrate Chemistry 37, 21-56 (2012)

Dalziel et al. Eur. J. Biochem. 271, 3623-3634 (2004)

Dennis et al. Science 236, 585-585 (1987)

Granowsky et al. Nat. Med. 6, 306-312 (2000)

Hedlund et al. Cancer Res. 68, 388-394 (2008)

Ihara et al. Glycobiology 14, 139-146 (2004)

Izawa et al. Cancer Res. 60, 1410-1416 (2000)

Lau et al. Cell 129, 123-134 (2007)

Lee et al. Mol. Cancer. Res. 6 1316-1325 (2008)

Lu et al. Mol. Cell Biochem. 122, 85-92 (1993)

Malagolini et al. Glycobiology 17, 688-697 (2008)

Miyagi et al. Proteomics 8, 3303-3311 (2008)

Ogata et al. Cancer Res. 55, 1869-1874 (1995)

Seales et al. Cancer Res. 65, 4645-4652 (2005)

Trinchera et al. Int. J. Biochem. Cell Biol. 43, 130-139 (2011)

Fabio Dall’Olio · 2012. 10. 26. · From: Trinchera et al. 2011 By immunoblot analysis, only a...

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