Mucins – their associated with carcinogenesis Dr Joy Burchell King's ...

Mucins – their associated with carcinogenesis 

Dr Joy Burchell 

King’s College London, UK 

Email: joy.burchell@kcl.ac.uk

Learning objectives 

•Understanding of the structure of Mucins 

•Understanding their glycosylation structure 

•To become familiar with their role in carcinogenesis 

Changes in expression 

Changes in glycosylation 

Relationship to tumour initiation and progression

Understanding of the structure of Mucins 

•They are the major glycoprotein in mucus and give 

mucus its viscosity. 

•They have a protective function, keeping unwanted 

; 

substances and micro-organisms at bay, while at the 

same time mediating specific interactions. 

•Mucins are large glycoproteins that are present at the 

interface between many epithelium and their external 

environment.


•Large molecular weight glycoproteins 

•More than 50% carbohydrate 

; 

•Oligosaccharide side chains are linked through oxygen 

to serine or threonine 

•Contain a domain of tandemly repeated amino acids rich 

in serine, threonine and proline. 

•Mucins can be either membrane bound or secreted


Have a general “bottle 

brush” structure 

Oligosaccharide 

; 

Polypeptide

Mucin Membrane/secreted Chromosome 

MUC1 M 1q21 

MUC2 S 11p15 

MUC3A/B M 7q22 

MUC4 M 3q29 

MUC5AC S 11p15 

MUC5B S 11p15 

MUC6 S 11p15 

MUC7 S 4q13-q21 

MUC8 S 12q24 

MUC11 M 7q22 

MUC12 M 7q22 

MUC13 M 3q13 

MUC15 M 11p14.3 

MUC16 M 17q21 

MUC17 M 7q22 

MUC18 M 

MUC19 S 12 

MUC20 M 3q21 

;


Sizes of tandem repeats 

MUC1 20 amino acids 


MUC3A/B 17 amino acids 


MUC5AC 8 amino acids 

; 

MUC5B 87 amino acids 




MUC11/12 28 amino acids 


MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC6, MUC7 all show allelic 

polymorphism. MUC5C does not.


Allelic variation 

MUC1, MUC2, MUC3A/B, MUC4, MUC5AC, MUC6, MUC7 

all show allelic polymorphism. 

MUC1: the number of tandem repeats in MUC1 can vary 

between 20-150 depending on the individual. 

M ; F 

Polymorphisms seen at the DNA, RNA and protein

Extracellular Membrane 

MUC2 

MUC5AC 

MUC5B 

MUC6 


MUC19 

MUC7 (monomer) 

MUC8 ? 

Epithelial mucins 

11p15 

Multi-mers 

; 

MUC1 

MUC3A 

MUC3B 

MUC4 

MUC12 

MUC11 

MUC13 

MUC15 

MUC16 

MUC17 

MUC18 

MUC20 

Selectin ligands: 

PSGL-1 

CD34 (GlyCAM- 

1)


Epithelial mucins 

Secreted Membrane 

MUC2 

MUC5AC 

MUC5B 

MUC6 

MUC19 

MUC7 (monomer) 

MUC8 

11p15 

; Multi-mers 

MUC1 

MUC3A 

MUC3B 

MUC4 

MUC12 

MUC13 

MUC15 

MUC16 

MUC17 

MUC18 

MUC20 

However, MUC1 has now been shown to be expressed 

on activated T cells and matured dendritic cells but the 

levels are low


;


Membrane mucins 

MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13 and 

MUC16 all appear to be post translationally cleaved into 2 

peptides in the ER. 

However, they stay together as they move through the Golgi 

and when the MUC is inserted into the membrane. 

O-linked oligossacharide 

Polypeptide 

; 

Cell membrane


MUC1 

; 

Taken from Kufe, Oncogene 2012:1-9


Secreted Mucins 

; 

N terminal D domains homologous to domains in VWF, rich in 

cystine, involved in intra and inter molecular bonds. 

C terminal C and CK cys-rich domains also have some homology 

to similar domains in VWF.


The structure of secreted mucins 

; 

Schematic representation of the secreted mucins MUC2, MUC5B, MUC5AC, 

and MUC6 localized on the chromosome 11 in p15.5. The domains D1, D2, D3, 

D', D4, B, C, and CK (cystin knot) show a high level of similarity with the 

respective domains of the pro-von Willebrand factor. The T/S/P domains are the 

domains rich in serine, threonine, and proline amino acid residues. Cys are 

domains rich in cystein amino acid residues, and PS is the peptide signal.


Assembly of secreted mucins 

1.Translation of the monomer 

2.The formation of dimers thro’ 

COOH CK domains and Nglycosylation. 

ER 

4. O-glycosylation of the dimer. 

5. The formation of multimers thro’ 

the NH 2 D-domains. Golgi. 

6.Translocation into secretory 

granules. 

7&8.Transport vesicles and mucin 

granules secreted by constitutive 

and regulatory exocytotic 

pathways.


Expression 

Membrane 

MUC1 most simple epithelial cells (activated T cells and DCs) 

MUC3A intestines, gall bladder and hepatocytes 

MUC3B intestines 

MUC4 trachea, bronchus, colon, breast 

MUC11/12 different tissues 

MUC16 ocular surfaces, cervical secretions, respiratory tract. 

Secreted 

MUC2 colon, small intestine, bronchus, trachea. 

MUC5AC stomach, respiratory tract 

MUC5B stomach, respiratory tract 

MUC6 stomach, colon, gallbladder, endocervix 

MUC7 sublingual salivary gland 

MUC8 tracheal, nasal 

;




•To become familiar with their role in carcinogensis 

; 




Mucin-type O-linked glycosylation 

•Sugars are added to serine and threonine and are 

linked through oxygen (O-linked). 

•No consensus peptide sequence for the addition of the 

sugars has been identified. 

; 

•Sugars are added singly and sequentially. 

•Each addition is catalysed by a glycosyltransferase. 

•The first sugar to be added is GalNAc. 

•Initiation of O-linked glycosylation occurs in the Golgi 

apparatus. 

•Several different “core” structures are formed, 

depending on the tissue, and these can be extended.


First sugar to be added N-acetylgalactosamine GalNAc 

UDPGalNAc + Thr/Ser-R 

UDP + GalNAc-Thr/Ser-R 

Large family of polypeptide GalNAcTs that can catalyse this reaction 

;


ppGalNAcTs 

•So far around 20 ppGalNAcTs have been cloned. 

•Distinct but overlapping specificities. 

•The tandem repeat of MUC1 has been used as a model 

㜰 

peptide to study the substrate specificity and kinetics of a 

number of ppGalNAcT. 

•Within each TR of MUC1 there are five potential sites for 

O-linked glycosylation. To get all the sites glycosylated 

require 3 ppGalNAcTs. 

•Some ppGalNAcTs required a glycosylated substrate

Structure of the MUC1 mucin 

Signal 

127aa 

Tandem repeat domain 

㜰 

HGVTSAPDTRPAPGSTAPPA 

The tandem repeat of MUC1, with five O-linked 

glycosylation sites has been used to determine the 

specificity of some polypeptide GalNAc-Ts 

TM


ppGalNAcTs can be distributed throughout the Golgi. 

Shown by tagging ppGalNAcTs with immuno-reactive 

epitope and EM using gold labelled second antibody. 

 

For review see: Gill, Clausen 

and Bard 2010 Location, 

location, location: new insights 

into O-GalNAc protein 

glycosylation. 

Trends in Biology

Basic structure of O-glycans 

Core Backbone Peripheral 

Lactosamine 

chains 

Terminal epitopes, 

αlinked

Common backbone structure on mucins 

Core 1 

Core2 

Core 3 

Core 4 

;

Formation of core 1 by β1,3GalT 

T synthase requires a molecular chaperone for 

activity 

ββββ1,3 GalT also known as T synthase because it catalyses the 

formation of core 1 also known as T or TF. 

 

(GalNAc Galβ1-3GalNAc) only GT that can catalyse this reaction 

Cosmc is located in the ER and is required for the correct folding and 

activity of T synthase in the Golgi. Private chaperone, only binds to T 

synthase. 

Dysfunction of Cosmc leads to the expression of the Ser/Thr-GalNAc 

also known as the Tn antigen 

J Biol Chem. 2010 Jan 22;285(4):2456-62.

Extension of the core structures





㿀 




Mucins and malignancy 

Change in the expression of mucins 

Mucins can be aberrantly glycosylated in 

㷰 

tumours resulting in: 

•Change of the number of sites 

•Change in the length of the sugar sidechains

Mucins and malignancy 

Mucins can be dramatically upregulated e.g MUC1 is 

upregulated 10-100 fold in breast carcinomas compared to 

the expression in the normal mammary gland. 

Ϛ

N 

Mucins can be over expressed in cancers 

MUC1 MUC5B MUC8 

C 

N 

េ◌ៀϜ 

MUC1, MUC5B and MUC8 expression in normal endometrial tissue 

(N) and endometrial cancers (Lau et al Am J Clin Pathol 2004;122:61-69) 

C 

N 

C

Changes in the glycosylation 

Change in the number of sites. 

•MUC1 purified from human milk has been shown to 

have, on average 2.5 sites glycosylated per tandem 

repeat. 

•MUC1 purified from a breast carcinomas cell line 

딀͵ 

has on average 4.5 sites glycosylated per tandem 

repeat. 

•Analysis of the sites occupied in other breast 

carcinoma cells shown a range from 2.5-5. This may 

reflect the the inability of ppGalNAcTs to get access 

to their site of glycosylation due to the length of 

adjacent chains that have already been 

glycosylated.

One mechanism may be the activation of Src, often seen in 

tumours, results in a relocation of ppGalNAcTs from the Golgi 

to the ER 

߰Ҕ 

This results in an increased density of glycosylation, increase in 

number of sites glycosylated 

Gill et. al. 2010 J. Cell Biol. 189, 843-858

Increased number of sites 

However other mechanisms may be related to the 

ditruption of the Golgi often seen in advance cancer 

cells 

and/or 

The decrease length of glycans on adjacent sites not 

߰Ҕ 

inhibiting the glycosylation of other sites 

Distribution of polypeptide 

GalNAcTs is seen 

throughout the Golgi

Changes in the length of the side chains 

Pathway of O-linked glycosylation of MUC1 in the 

mammary gland 

T epitope 

Tn epitope 

C2GnTI 

β1,3GalT 

Galβ1,3GalNAc 

β1,6 

GlcNAc 


ppGalNAcTs 

Galβ1,3GalNAc 

GalNAc-αSer/Thr 

ϝ 

ST3Gal-I 

SAα2,3Galβ1,3GalNAc 


α2,6 

SA 

ST6GalNAcI 

STn epitope 

SAα2,6GalNAc 

ST epitope

α3 

α6 

α 

Sialyl-Tn 

β3 

α 

Sialyl-T 

S/T 

S/T 

Cancer Cell 

ST6GalNAc I 

Tn 

Peptide 

α 

S/T 

Normal Cell 

ST3Gal -I α * C2GnT 

α S/T 

β3 

Sialylation Sialylation, 

elongation 

Core 1 

ppGAT 

C1GalT 

S/T 

β6 

β3 

β4GalT, 

sialylation, 

elongation 

GalNAc Gal GalNAc Neu5Ac 

Core 2

SM3 HMFG2 

泐͵

Change in O-linked glycans attached to MUC1 in breast 

cancer: SM3 binding is blocked by long O-glycans 

SM3 

-ve 


SM3 

Common event as 90% breast carcinomas react with SM3: 

Must be advantageous to the tumour in some way? 

ͺ 

Malignancy 

SM3 

+ve

Mechanisms responsible for the changes in glycan 

side chains 

Mutations in cosmc 

Changes in the expression of glycosyltransferases 

洠͵

Mechanisms responsible for the aberrant O-linked 

glycosylation. 

Mutations in Cosmc results in the expression of Tn and if 

ST6GalNAc-T expressed STn 

Ju, T. et al. Cancer Res 2008;68:1636-1646 

Copyright ©2008 American Association for Cancer Research 

ͺ

Dysfunctional Cosmc is not the main mechanism 

for Tn expression in breast cancer 

浰͵

Activation of Src, often seen in tumours, results in a 

relocation of ppGalNAcTs from the Golgi to the ER 

ͺ 

This results in an increased density of glycosylation, increase in 

number of sites glycosylated 

Gill et. al. 2010 J. Cell Biol. 189, 843-858 

As high density of O-GalNAc may hinder T synthase and C2GnT – 

result in shorter side chains and expression of Tn, STn or core 1

Mechanisms responsible for the aberrant Olinked 

glycosylation. Changes in expresssion of 

glycosyltransferases 1 

Ser/Thr-GalNAc SAα2,6GalNAc 

The sialyl Tn epitope 

Expressed by about 30% of breast cancers 

100% correlation between the expression of the glycan 

and the glycosyltransferase ST6GalNAc-I 

(Sewell et al 2005 JBC) 

淀͵ 

Expression is the result of turning on of the sialyltransferase 

ST6GalNAc-I

In breast carcinomas, expression of STn correlates with 

ST6GalNAc-1 expression 

BrCa Case Sialyl Tn Northern RT-PCR 

13 Negative Negative Negative 

3 Negative Negative Not analysed 


26 Weak Positive Negative Positive 






28 Positive Positive Not analysed 


5 

23 

Negative 

Weak Positive 

Negative 

ͺ 

Negative 

Negative 

Positive 

19 Negative Negative Not 










1 Positive Negative Positive 



10 Positive Positive Positive 

24 Positive Positive Positive 

25 Negative Negative Negative

Expression of ST6GalNAc-I over-rides existing Oglycosylation 

in T47D cells 

 

Myc 

ST6GalNAc-1 

Anti-STn

Expression of ST6GalNAc-I over-rides existing Oglycosylation 

in MTSV1-7 cells that express core 2 

glycans 

Myc tagged ST6GalNAc-I STn expression 

暀͵ 

Infers located early in the Golgi – mapping by electronmicroscopy 

using tagged ST6GalNAc- I and immuno-gold shows the GT is 

located throughout the Golgi

Mechanisms responsible for the aberrant Olinked 

glycosylation Changes in expresssion 

of glycosyltransferases 

The sialyl T epitope: SAα2,3 Galβ1,3GalNAc 

Common O-glycan, often found in normal cells 

But not found on MUC1 when it is expressed by normal cells 

Increased expression results from an over-expression of 

ST3Gal-I

ST3Gal-I mRNA expression by breast tissues (in situ 

hybridisation) 

ͺ

ST3Gal-I expression is up-regulated and associated with 

grade 

Benign Grade I 

Grade III 

SCORE % AND INTENSITY 

6 

5 

4 

3 

2 

1 

0 

BENIGN 

1 

Grade I Grade II Grade III

ST3Gal-I and C2GnT1 compete for a common 

substrate 

Galβ1-3GalNAc 

ST3Gal-I C2GnT1 

SAα2,3 Galβ1-3GalNAc Galβ1-3GalNAc 

β1,6 

GlcNAc 

쉐Ϥ

What is the relevance of over-expression of 

mucins and the aberrant O-glycosylation? 

Immunotherapy 

Cancer-associated antigenically distinct 

Induce a humoral response 

Change in glycosylation could alter uptake by and 

breakdown by APCs. Through lectin interactions? 

ͺ 

Can be used as target for T cells that express chimeric 

antigen receptors (CAR) 

Used as a biomarker? 

Biological relevance to the tumour? 

Very common event 

Murine tumours expressing human MUC1 carrying sialyl T 

grow faster than tumours expressing MUC1 carrying core 2 

glycans in MUC1 TG mice





쉐Ϥ 




What is the relevance of over-expression of 

mucins and the aberrant O-glycosylation? 

Immunotherapy 

Cancer-associated antigenically distinct 

Induce a humoral response 

Change in glycosylation could alter uptake by and 

breakdown by APCs. Through lectin interactions? 

ͺ 

Can be used as target for T cells that express chimeric 

antigen receptors (CAR) 

Used as a biomarker? 

Biological relevance to the tumour? 

Very common event 

Murine tumours expressing human MUC1 carrying sialyl T 

grow faster than tumours expressing MUC1 carrying core 2 

glycans in MUC1 TG mice

Cancer cells lose their polarity 

Carraway et al Future Oncol. 2009 December; 5(10): 1631–1640. 

ͺ

Loss of cell polarity in Cancer 

Upregulation of MUC1 and loss of cellular polarity allows 

interaction with EGFR leading to increased signaling 

쉐Ϥ

Changes in glycosylation of MUC1 in breast cancer 

Increased expression of ST3Gal-I can promote tumorigenesis 

Picco et al 2010 Glycobiology 

ͺ

MUC1 carrying a tumour associated glycan can interact 

with galectin 3 

TF or T antigen Galβ1,3GalNAc 

Galectin 3 - one of the galectin family of galactose binding proteins 

Concentration of galectin 3 increased in the sera of breast cancer patients 

(Yu et al 2007 J Biological Chemistry 283:773-781) 

ͺ

MUC1 carrying sialylated tumour associated glycans 

could interact with Siglecs 


Siglecs are lectins on the surface of cells of the immune 

system that bind sialic acid 

װ 

MUC1 has been shown 

to bind to Siglec1 

(sialoadhesin) 

Nath et al Immunology, 

1999, 98: 213-219 and 

Siglec 3 (unpublished 

results).

Aberrant glycosylation of MUC1 may enhance 

uptake by lectins on APCs 

MUC1 carrying Tn tumour associated glycan can interact with MGL 

MGL is a lectin expressed on immature dendritic cells that can bind GalNAc 

MGL transfected cells Dendritic cells 

װ 

(Napoletano et al 2007 Journal of 

Immunology, 67:8358-8367)

However things are not also ways that simple!!!! 

Around 75% of breast cancers are defined as ER-positive. 

This means they express the estrogen receptor and the 

hormone estrogen drives the proliferation of the tumour. 

Around 25% are therefore ER negative and these seem to 

have have a different pathway of mucin glycosylation 

װ

Mucin glycosylation of ER positive and ER 

negative breast cancers 

ER+ve 

tumours 

ER-ve 

tumours 

α2-3 

β1-3 

Expression of SLe x 

α 

R 

ST3Gal-I 

fucosyltransferases and 

sialyltransferase 

β1-6 α 

β1-3 

Core 1 

R 

β1-3 

װ 

α 

β1-6 α 

β1-3 

α 

R 

R 

C2GnT1 

R 

C2GnT1 

Core2 

R 

Core2 

β1-6 α 

β1-3 

Elongation 

from the 

Gal and 

GlcNAc 

GalNAc Gal GlcNAc Neu5Ac 

R 

Normal 

mammary 

epithelium 

ER-ve tumors are also more likely to carry more sLe X antigen

Analysed sLe x expression by Breast cancers 

352 consecutive primary human breast carcinomas were stained for the 

expression of sLe x (antibody HECA-452) 

Julien et al Cancer Res 2011 Cancer Research 

װ

But it is even more complicated as some ER+ve can 

express sLex!! 

Julien et al Cancer Res 2011 

װ

sLe x 

Changes in glycosylation may promote metastasis 

E-selectin is constitutively 

expressed in the 

microvasculature of bone 

marrow 

Taken from Sackstein 2009 Immunological Reviews Vol. 230: 51–74 

װ

Within the ER positive cancers high levels of sLex 

expression was associated with bone metastasis 

ER+ve ER-ve 

p=

Changes in mucin glycosylation and 

the affects on tumour progression is 

complicated!! 

װ

From this lecture it is hope that you now know 

•The basis of the structure of Mucins 

•The glycosylation of MUC1 in the normal mammary gland and breast 

carcinomas 

•Have an idea of the role of mucins in carcinogenesis 



Relationship to tumour initiation and progression 

װ

Thanks you for listening!!! 

װ

Mucins – their associated with carcinogenesis Dr Joy Burchell King's ...

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