Mucins: Structural diversity, biosynthesis, its role in pathogenesis and as possible therapeutic targets

Critical Reviews in Oncology / Hematology 122 (2018) 98–122

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Mucins: Structural diversity, biosynthesis, its role in pathogenesis and as possible therapeutic targets Suresh Sulekha Dhanisha1, Chandrasekharan Guruvayoorappan Prathapan Abeesh1

T

⁎,1

, Sudarsanan Drishya1,

Laboratory of Immunopharmacology and Experimental Therapeutics, Division of Cancer Research, Regional Cancer Centre, Medical College Campus, Thiruvananthapuram 695011, Kerala, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Secreted mucins Transmembrane mucins Pathogenesis Therapeutic agents

Mucins are the main structural components of mucus that create a selective protective barrier for epithelial surface and also execute wide range of other physiological functions. Mucins can be classified into two types, namely secreted mucins and membrane bounded mucins. Alterations in mucin expression or glycosylation and mislocalization have been seen in various types of pathological conditions such as cancers, inflammatory bowel disease and ocular disease, which highlight the importance of mucin in maintaining homeostasis. Hence mucins can be used as attractive target for therapeutic intervention. In this review, we discuss in detail about the structural diversity of mucins; their biosynthesis; its role in pathogenesis; regulation and as possible therapeutic targets.

1. Introduction The mucus is a complex viscoelastic adherent secretion of goblet cells in the vascular epithelium, which lines all organs that are in contact with the external environment including gastrointestinal, respiratory, reproductive tract and ocular surface. It is mainly involved in lubrication, maintaining hydration and acts as a barrier against entry of pathogens and other harmful substances to the epithelium. This secretion which plays a major role in innate immune response, is composed of water, salts and a series of proteins and glycoproteins interwoven together to form a viscoelastic product (Bansil and Turner, 2006). Among these, mucin family of glycoproteins contribute to mucoadhesive, hydrophobicity and viscoelastic nature of mucus, thereby protecting epithelium from chemical, enzymatic and mechanical damage. The mucoadhesive nature of mucins helps them to adhere to other substances through hydrogen bonds, hydrophobic and electrostatic interactions, resulting in the formation of gel aggregates. Based on the structure and localization, mucins can be classified into two types namely secreted mucins and membrane bounded (transmembrane) mucins. Secreted mucins usually form a protective layer over organs that are in contact to the external environment and creates a physical barrier against pathogens. Membrane bound mucins on the other hand, are retained in plasma membrane due to the

presence of a hydrophobic membrane spanning (transmembrane) domain and they have role in various signalling pathways. Both types of mucins have attracted scientific community and have made possible to a greater understanding of their pathophysiological role which in turn have given recent attention towards these complex molecules and their potential as biological tools for improved treatments in future. In the review, we discuss about this family of large glycoproteins, their structural diversity, biosynthesis, pathophysiological role and its possible application as a potential marker for disease diagnosis. 2. The mucin family Mucins belong to the family of large glycoproteins that constitute the major structural component of mucus. Mucins are encoded by mucin genes, represented as MUC in humans followed by a number that reflects the order in which the particular mucin gene was discovered. The following mucin genes have been identified in humans till dateMUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC19, MUC20, MUC21 and MUC22. Mucin gene is translated into a rod shaped apomucin which is further decorated with extensive glycosylation. Mucin glycoprotein is composed of 80% carbohydrates and 20% protein core. The carbohydrate moieties primarily

⁎ Corresponding author at: Laboratory of Immunopharmacology and Experimental Therapeutics, Division of Cancer Research, Regional Cancer Centre, Trivandrum 695 011, Kerala, India. E-mail addresses: [email protected], [email protected] (C. Guruvayoorappan). 1 All the authors deserve equal contribution in this manuscript.

https://doi.org/10.1016/j.critrevonc.2017.12.006 Received 16 June 2017; Received in revised form 28 October 2017; Accepted 12 December 2017 1040-8428/ © 2017 Elsevier B.V. All rights reserved.

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Table 1 Classification of human mucins. Mucin Genes

Membrane Bound Mucins MUC1

Chromosome Locus

Distribution

References

1q22

Stomach, breast, gall bladder, cervix, pancreas, respiratory tract, colon, duodenum, oesophagus, kidney, eye, B cell, T cell, middle ear epithelium Small intestine, colon, gall bladder, duodenum, middle ear epithelium Small intestine, colon, gall bladder, duodenum, middle ear epithelium Respiratory tract, stomach, cervix, colon, eye, middle ear epithelium Colon, small intestine, stomach, pancreas, lung, kidney, prostate, uterus Colon, small intestine, trachea, kidney, appendix, stomach, middle ear epithelium Heart, kidney, lungs Spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone marrow, lymph node, tonsil, breast, fetal liver, lungs, middle ear epithelium Peritoneal mesothelium, reproductive tract, respiratory tract, eye, middle ear epithelium Small intestine, colon, duodenum, stomach, middle ear epithelium Kidney, stomach, duodenum, placenta, colon, lungs, prostate, liver, middle ear epithelium Lungs, large intestine, thymus and testis Lungs, placenta, testis

(Andrianifahanana et al., 2001; Arul et al., 2000; Brayman et al., 2004; Buisine et al., 2000; Copin et al., 2001; Gendler and Spicer, 1995; Guillem et al., 2000; Ho et al., 1995a; Lan et al., 1990) (Gum et al., 1990)

Small intestine, cell, eye, middle ear epithelium. respiratory tract, colon Respiratory tract, stomach, cervix, eye, middle ear epithelium Respiratory tract, salivary gland, cervix, gallbladder, pancreas, seminal fluid, middle ear epithelium Stomach, duodenum, gallbladder, pancreas, seminal fluid, cervix, middle ear epithelium Sublingual gland, submandibular gland, respiratory tract, eye, middle ear epithelium

(Audie et al., 1993; Buisine, 1999; Buisine et al., 2000; Copin et al., 2001; Gum et al., 1994) (Buisine, 1999; Gipson and Inatomi, 1997; Porchet et al., 1995)

Salivary gland, respiratory tract, middle ear epithelium Trachea, cervix, testis, endometrium, placenta Oviduct

(Bobek et al., 1993) (D’Cruz et al., 1996; Shankar et al., 1994) (Arias et al., 1994)

MUC3A

7q22.1

MUC3B

7q22

MUC4

3q29

MUC12

7q22

MUC13

3q21.2

MUC14/Endomucin MUC15

4q24 11p14.3

MUC16

19p13.2

MUC17

7q22

MUC20

3q29

MUC21/epiglycanin MUC22 Secreted Mucins Gel-forming mucins MUC2

6p21.33 6p21.33

11p15.5

MUC5AC

11p15.5

MUC5B

11p15.5

MUC6

11p15.5

MUC19

12q12

Non-gel-forming mucins MUC7 MUC8 MUC9/OVGP1 (Oviductal glycoprotein1)

4q13.3 12q24.33 1p13.2

(Pratt et al., 2000) (Gipson and Inatomi, 1997; Porchet et al., 1995) (Moehle et al., 2006; Williams et al., 1999) (Williams et al., 2001) (Liu et al., 2001) (Pallesen et al., 2002; Shyu et al., 2007)

(Argueso et al., 2003; Matsuoka et al., 1990; Yin and Lloyd, 2001; Zeimet et al., 1998) (Gum et al., 2002; Moehle et al., 2006) (Higuchi et al., 2004) (Itoh et al., 2008) (Hijikata et al., 2011)

(Audie et al., 1995; Audie et al., 1993; Balague et al., 1994; Campion et al., 1995) (Gipson et al., 1997; Ho et al., 1995b; Toribara et al., 1997) (Chen et al., 2004)

an amino terminal extra cellular region densely decorated with glycans and a carboxy terminal intracellular cytoplasmic tail. The extracellular domain of transmembrane mucins is mainly composed of variable number of tandem-repeat (TR) domain, SEA (Sea urchin sperm protein, enterokinase, and agrin) domain or epidermal growth factor (EGF)-like domain. Instead of SEA domain, MUC4 contains other three domains namely NIDO (nidogen-like domain), AMOP (adhesion-associated domain in MUC4 and other proteins) and vWD (Von Willebrand factor type D domain) (Duraisamy et al., 2006) (Fig. 1). Tandem repeats are heavily glycosylated regions rich in serine, threonine and proline residues and are characteristic of mucin core proteins. It may be highly polymorphic for length and sequence variability and is poorly conserved and repeated multiple times. The number of repeats and sequence varies in each family member. SEA domain is highly conserved and consists of approximately 100 amino acid residues lying close to the membrane on the luminal side. This domain was initially identified in MUC1 and is found to have role in protein glycosylation. The SEA domain has an auto proteolytic cleavage site that separates the mucin into two subunits: a larger extracellular subunit made of variable number of tandem repeats and a smaller subunit containing short extracellular domain with SEA module and EGF-like domain, single transmembrane domain and cytoplasmic tail. Both subunits are noncovalently linked after the cleavage (Macao et al., 2006). Epidermal growth factor (EGF)-

attached to the mucin protein core includes N-acetylgalactosamine, Nacetylglucosamine, galactose, fucose, sialic acid (N-acetylneuraminic acid) and traces of sulfate and mannose. The peculiar feature of mucin glycoproteins is the presence of tandem repeat structures with high proportion of proline, threonine and serine, that forms the PTS domain. The PTS domain is extensively O-glycosylated through N-acetylgalactosamine O-linkages (GalNAcO-linkages) at the threonine and serine residues. The carbohydrate residues are present in a “bottle brush” configuration around the protein core. 2.1. Mucin types Mucin glycoproteins are sub-classified into two structurally different groups: the secreted and the membrane-bound mucins (Transmembrane mucins). The different types of human mucins, their genes, chromosome locus and distribution are demonstrated in Table 1. 2.1.1. Membrane bound mucins The following transmembrane mucins have been identified in humans till date: MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, EMCN or MUC14, MUC15, MUC16, MUC17, MUC20, MUC21 and MUC22. Transmembrane mucins are type-I membrane anchored proteins with a single membrane spanning domain anchored to the plasma membrane, 99

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Fig. 1. Schematic representation of transmembrane mucin along with unique domains in Mucin 4. Transmembrane mucins are type-I membrane anchored proteins with a single membrane spanning domain anchored to the plasma membrane, an amino terminal extra cellular region densely decorated with glycans and a carboxy terminal intracellular cytoplasmic tail. The extracellular domain is mainly composed of variable number of tandem repeat (TR) domains, SEA (sea urchin sperm protein, enterokinase, and agrin) domain or epidermal growth factor (EGF)-like domain. Instead of SEA domain, MUC4 contains other three domains namely NIDO (nidogen-like domain), AMOP (adhesion-associated domain in MUC4 and other proteins) and vWD (Von Willebrand factor type D domain).

from MUC5B secreted mucin (Duraisamy et al., 2007). Nuclear Magnetic Resonance (NMR) studies have revealed that Mucin 1 mainly contains random coil, small amount of beta sheets and very little alpha helix (Fontenot et al., 1993; Otvos and Cudic, 2003). A 200 KDa of mucin 1 protein is comprised of 20 amino acids residue tandem repeat, an SEA domain, a transmembrane domain and 69 amino acids long C-terminal cytoplasmic tail (Fig. 2). The cytoplasmic domain has several phosphorylation sites that have role in signal transduction and might interact with cytoskeletal elements (Hattrup and Gendler, 2008; Lan et al., 1990; Singh and Hollingsworth, 2006). The core protein of mucin 1 has a molecular weight of 120–225 KDa whereas the mature glycosylated protein has a molecular weight range of 250–500 KDa (Gendler and Spicer, 1995; Gendler et al., 1991; Lancaster et al., 1990). Mucin1 is a heterodimer. After translation of MUC1 gene, auto proteolysis of the single polypeptide occurs at the SEA domain to obtain two subunits namely MUC1N (MUC1 N-terminal) and MUC1C (MUC1 C-terminal) (Levitin et al., 2005; Ligtenberg et al., 1992; Macao et al., 2006). The MUC1N contains highly glycosylated and conserved tandem repeats of 20 amino acids (Gendler et al., 1988; Siddiqui et al., 1988). Mucin1 N-terminal forms a stable non covalent complex with MUC1C by which it is anchored to the cell surface. After the release of MUC1N component of the mucin-1 heterodimer from the cell surface, MUC1C is left as a putative receptor that has diverse role in signaling pathways (Kufe, 2008). Mucin1 is normally localized to the apical membranes of normal secretory epithelial cells (Kufe et al., 1984). During transformation and loss of polarity, mucin1 has shown to be overexpressed on the entire surface of diverse types of carcinoma cells. The splice variants of MUC1 include Variant CT58, Variant CT80, Variant SEC, Variant X, Variant Y and Variant ZD. The MUC1-Y isoform lacks the tandem repeat region.

like domain shows homology to epidermal growth factor and other related growth factors and cytokines. It mediates interaction between mucin subunits and ErbB (erythroblastosis oncogene B) receptors. The number of amino acid residues in the cytoplasmic tail varies among different mucins (22 amino acid residues in MUC4 to 80 amino acids in MUC17). The cytoplasmic tails are relatively divergent. MUC1 has a longer cytoplasmic tail with numerous tyrosine phosphorylation sites (Hattrup and Gendler, 2008). While MUC3, MUC12 and MUC17 have PDZ [post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1)] binding motifs in their far C-terminal region (Malmberg et al., 2008). The cytoplasmic tail interacts with cytoskeleton. The key feature that differentiates transmembrane mucins from other membrane bound glycoproteins is the presence of centrally located tandem repeat regions. The different transmembrane mucins are described below. 2.1.1.1. Mucin 1. Mucin 1 is a type I transmembrane mucin that is expressed at a basal level in most epithelial cells (Patton et al., 1995). Mucin 1 is normally expressed in airways (Copin et al., 2001), salivary glands (Liu et al., 2002), breast (Gendler and Spicer, 1995), oesophagus (Arul et al., 2000; Guillem et al., 2000), stomach (Ho et al., 1995a), pancreas (Andrianifahanana et al., 2001; Buisine et al., 2000), duodenum (Buisine et al., 2000), male and female reproductive tract (Brayman et al., 2004). MUC1 is also known as episialin, polymorphic epithelial mucin (PEM), peanut lectin-binding urinary mucin, epithelial membrane antigen (EMA), DF3 antigen (recognized by DF3 monoclonal antibody) and HMFG (human milk fat globule) antigen. MUC1 gene is located on human chromosome locus 1q22. It was the first mucin gene to be cloned (Batra et al., 1992; Batra and Hollingsworth, 1991; Batra et al., 1991; Lan et al., 1990). It has been found that MUC1 evolved 100

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Fig. 2. Structural diversity of different transmembrane mucins. The figure illustrates domain structures of different transmembrane mucins. The tandem repeat region is rich in proline, threonine and serine (PTS domain) which is heavily glycosylated and forms the mucin-like domain. SEA, sea urchin sperm protein, enterokinase, and agrin domain; EGF-like, Epidermal Growth Factor-like domain; NIDO, Nidogen-like domain; vWD, Von Willebrand Factor Type D domain; AMOP, adhesion-associated domain in MUC4 and other proteins; PI3K-RBD, Phosphatidylinositol 3-kinase RAS-binding domain; ANK, Ankyrin repeats.

site to generate two subunits- MUC4α and MUC4β. MUC4α, the mucin type subunit, is the large extracellular subunit with a molecular weight of 850 kDa. This subunit is rich in serine, threonine and proline residues and is heavily glycosylated. MUC4α contains three putative functional domains like tandem repeat domain with 16 amino acid residues repeated up to 400 times, nidogen-like domain (NIDO) and adhesionassociated domain in MUC4 and other proteins (AMOP). It also contains sequences that have high degree of similarity with Von Willebrand factor type D domain (vWD), but the cysteine residues that are characteristic of this domain are not conserved in MUC4. The GDPH cleavage site is located in vWD domain. AMOP with eight invariant cysteine residues has been shown to mediate adhesion. NIDO domain is similar to the nidogen-EGF domain of ancestral nidogen proteins. It facilitates metastasis of pancreatic cancer cells (Senapati et al., 2011). MUC4β is the transmembrane subunit with molecular weight of 80 kDa that contains two domains rich in N-glycosylation sites, three EGF-like domains, a transmembrane domain and 22 amino acids long cytoplasmic tail (Chaturvedi et al., 2008; Duraisamy et al., 2006) (Fig. 2). This subunit is considered as an oncogene due to its involvement in signaling pathways (Albrecht and Carraway, 2011). The glycosylated alpha subunit provides anti-adhesive properties to the cell, aiding in cell-cell and cell-matrix detachment. Due to the presence of variable number of tandem repeats, MUC4 in humans is highly polymorphic in nature (Debailleul et al., 1998). 24 different splice variants of MUC4 have been identified in normal and malignant human tissues. Among these, 22 variants are produced by alternate splicing of the 3′ terminal exons and are known as sv1 to sv21-MUC4. The remaining two splice variants are created by alternative splicing of exon 2 and are named as MUC4/X and MUC4/Y. sv0-MUC4 is the main isoform that encodes full length MUC4 protein (Choudhury et al., 2000a, 2000b; Moniaux et al., 2000).

2.1.1.2. Mucin 3A. Mucin 3A is an intestinal membrane bound mucin encoded by MUC3A gene residing at chromosome locus 7q22.1 (Fox et al., 1991; Fox et al., 1992; Gum et al., 1990). It consists of two tandem repeat domains, one with 375 amino acid residues and the second with 17 amino acid residues tandem repeats (Gum et al., 1997) at the amino terminus of the extracellular domain, an SEA module flanked by two cysteine rich EGF-like domains, a transmembrane domain and 72 amino acids long cytoplasmic tail at the carboxy terminal (Crawley et al., 1999; Guddo et al., 1998) (Fig. 2). 2.1.1.3. Mucin 3B. Mucin 3B is also an intestinal cell surface associated mucin encoded by MUC3B gene located at the chromosome locus 7q22. Both MUC3A and MUC3B have evolved from the same ancestral gene which has undergone gene duplication and allelic variations in the chromosome 7q22 (Pratt et al., 2000). Pratt et al. has shown that 95% of the exonic and intronic sequences at the carboxy termini are identical in MUC3A and MUC3B. MUC3A and MUC3B contain 91% conserved amino termini. Both these proteins are structurally similar, possessing tandem repeat domain, 2 EGF-like domains, a transmembrane domain and a cytoplasmic tail (Fig. 2). Studies have shown that Mucin 3B contains at least 11 exons and the tandem repeat region has same amino acid sequence like that of mucin3A, but with more substitutions. Studies have detected MUC3B expression in small intestine, colon and Caco-2 cells (Pratt et al., 2000). 2.1.1.4. Mucin 4. Mucin 4 is a cell surface associated mucin encoded by MUC4 gene and is localized to chromosome locus 3q29 (Gross et al., 1992). Mucin 4 expression has been detected in bronchus, endocervix, prostate (Porchet et al., 1995) and conjunctival epithelium (Gipson and Inatomi, 1997), with weak expression in colon, gastric epithelium and small intestine (Porchet et al., 1995). MUC4 is also expressed by the epithelial surface of the oral cavity, eye, lachrymal glands, middle ear, salivary glands, female reproductive tract, prostate gland, lungs and mammary glands to protect and lubricate these surfaces. Unlike other transmembrane mucins, mucin 4 lacks the SEA domain. MUC4 is translated into a single polypeptide chain of molecular weight 930 KDa that is cleaved at the GDPH (Gly-Asp-Pro-His) proteolytic cleavage

2.1.1.5. Mucin 12. Mucin 12 is a transmembrane mucin encoded by MUC12 gene that resides on the human chromosome 7 in the region q22.1. MUC12 expression has been detected in major organs including colon, pancreas, prostate and uterus (Moehle et al., 2006; Williams et al., 1999). MUC12 is translated into a 585 amino acid long 101

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cytoplasmic tail with 76 amino acid residues (Pallesan et al., 2002; Shyu et al., 2007) (Fig. 2).

polypeptide that consists of an amino terminal mucin-like tandem repeat domain of 28 amino acid residues rich in serine/threonine and proline, 150 amino acids long non-mucin-like domain flanked on either side by cysteine rich EGF-like domains, a transmembrane domain and 75 amino acids long cytoplasmic domain at the carboxy terminal (Fig. 2). The non-mucin-like domain possesses five N-glycosylation sites and a potential coiled-coil domain. The cytoplasmic tail contains YNNF (Tyr-Asn-Asn-Phe) sequence (amino acids 557–560) that can be recognized by SH2 domain-containing proteins (Songyang et al., 1994), suggesting the role of MUC12 in signal transduction (Williams et al., 1999).

2.1.1.9. Mucin 16. MUC16 is the largest transmembrane mucin with a molecular weight ranging from 2.5 to 5 million Dalton (O’Brien et al., 2001; O'Brien et al., 2002). MUC16 resides in the chromosome locus 19p13.2 (Yin and Lloyd, 2001). Studies have detected its expression in epithelium of upper respiratory tract (Matsuoka et al., 1990), ocular surface (Argueso et al., 2003), mesothelium lining body cavities such as pleural, peritoneal, and pelvic cavities, internal organs and male and female reproductive organs (Zeimet et al., 1998) to protect and lubricate these surfaces. The mucin16 protein consists of an amino terminal region, an extensive tandem repeat domain, and a carboxy terminal region with a short cytoplasmic domain. The N-terminal domain is made of 12,070 amino acids rich in serine or threonine and has multiple O-glycosylation sites. The tandem repeat domain consists of 156 amino acids long sequences repeated 18–60 times. The carboxy terminal domain consisting of 284 amino acids is further divided into three distinct domains such as an extracellular domain, a transmembrane domain and a cytoplasmic tail (O’Brien et al., 2001; O'Brien et al., 2002). The extracellular domain possesses many Nglycosylation sites and a few O-glycosylation sites (Kui et al., 2003). Unlike other transmembrane mucins, human mucin16 has 56 SEA modules and it lacks EGF-like domain. The SEA module is followed by a transmembrane domain of 21 amino acid residues. The cytoplasmic tail is made of 32 amino acid residues and it has many phosphorylation sites (Fig. 2). MUC16 has four leucine-rich repeats and two ANK (Ankyrin) repeats (Yin and Lloyd, 2001). The cytoplasmic tail has polybasic amino acid sequence that interacts with the cytoskeleton through ERM (ezrin/radixin/moesin) actin-binding proteins. MUC16 has been recognized as a tumor-associated antigen which is cleaved from ovarian cancer cell surface into the blood stream. Hence MUC16 is considered as a well-known biomarker for ovarian cancer. CA125 is identified as a peptide epitope of MUC16 (O’Brien et al., 2001; Yin and Lloyd, 2001), which is found to promote cancer cell proliferation as well as inhibits ant-cancer immune response (Bast and Spriggs, 2011; Rump et al., 2004).

2.1.1.6. Mucin 13. Mucin 13 is a cell surface associated mucin that resides on the chromosome locus 3q21.2. In normal human tissues MUC13 expression has been detected in trachea, oesophagus, gastric epithelium, small intestine, large intestine and kidney (Williams et al., 2001). It consists of an amino terminal signal sequence followed by ten tandem repeats, each containing 151 amino acids. This domain is rich in O-glycosylated serine, threonine and proline which is crucial for maintaining the structure and function of mucin. The central region contains three epidermal growth factor (EGF)-like domains namely EGF1, EGF2 and EGF3, which have role in signalling pathway. In between EGF1 and EGF2-like domains, a SEA module is present which provides a proteolytic cleavage site that separates MUC13 into two subunits- an extracellular alpha (α) subunit and a transmembrane beta (β) subunit. The SEA domain may be cleaved during transport of MUC13 to the cell surface, after which α and β subunits are covalently linked. A short transmembrane domain is present adjacent to the EGF3like domain. The carboxy terminal contains cytoplasmic domain with 69 amino acid residues (Williams et al., 2001) (Fig. 2). Studies have revealed the anti-apoptotic and anti-inflammatory effects of MUC13 in epithelial cells (Sheng et al., 2013; Sheng et al., 2011). Under normal physiological conditions, MUC13 perform the role of protecting the epithelial surfaces of gastrointestinal tract, respiratory tract and reproductive tract. MUC13 has been found to be aberrantly expressed in many cancers (Chauhan et al., 2009b; Maher et al., 2011; Shimamura et al., 2005).

2.1.1.10. Mucin 17. Mucin 17 is a membrane bound mucin (Moniaux et al., 2006) encoded by MUC17 gene that is located on the human chromosome 7 in the region q22 (Gum et al., 2002). MUC17 expression has been detected in duodenum, colon (Gum et al., 2002) and terminal ileum (Moehle et al., 2006). Mucin 17 glycoprotein is 4493 amino acids long consisting of a signal sequence, a large amino terminal domain containing a central tandem repeat region of 59 amino acids that is repeated 63 times, two EGF-like domains flanking a SEA module, a transmembrane domain and a carboxy terminal domain having an 80 amino acids long cytoplasmic tail possessing phosphorylation sites for serine and tyrosine. The central tandem repeat domain is followed by a unique degenerate tandem repeat region and mucin-like sequences rich in serine, threonine and proline amino acids. The carboxy terminal possesses sites for N-glycosylation. The tandem repeat region of MUC17 shows a low degree of polymorphism. MUC17SEC is a splice variant of MUC17 that encodes a truncated protein lacking EGF2-like domain, transmembrane domain and cytoplasmic tail (Fig. 2). Since it is devoid of transmembrane domain, MUC17SEC is considered as a soluble protein (Moniaux et al., 2006). In the epithelial cells MUC17 functions to provide signal transduction, maintain luminal structure, provide cytoprotection and gives anti-adhesive properties to cancer cells that lose their polarity. Studies have shown that MUC17 is deregulated in pancreatic cancer (Moehle et al., 2006; Moniaux et al., 2006). MUC17 also function in maintaining the integrity of intestinal epithelium.

2.1.1.7. Mucin 14/endomucin. Endomucin is also known as endothelial sialomucin, EMCN, mucin 14 or mucin-like sialo glycoprotein. It is a transmembrane mucin rich in sialic acid and its gene MUC14 is located on the human chromosome locus 4q24. EMCN has been found to be highly expressed in vascular tissues like heart, lungs and kidney. The endothelial cells that line post capillary venules express MUC14. These are the primary site of leukocyte recruitment during inflammation (Liu et al., 2001). Endomucin is made of 261 amino acids with a molecular weight of 27.5 kDa and contains an N-terminal signal sequence, uteroglobin homology domain, a phosphatidylinositol 3-kinase RASbinding domain (PI3K-RBD), a transmembrane domain and C-terminal cytoplasmic domain (Fig. 2). It has several sites for phosphorylation, Nglycosylation and N-myristoylation. It can exist both as an unprocessed precursor peptide as well as a processed protein of 241 amino acid residues known as endomucin-2. EMCN doesn’t contain tandem repeats in its extracellular domain, but it has several extracellular glycosylated serine and threonine residues. Studies have shown that MUC14 inhibits cell and extracellular matrix interaction. Endomucin is found to inhibit leukocyte—endothelial cell adhesion (Zahr et al., 2016). 2.1.1.8. Mucin 15. Mucin 15 is a cell surface associated mucin encoded by MUC15 gene located in the human chromosome locus 11p14. It was initially isolated from bovine milk fat globule membranes (Pallesen et al., 2002). Studies have detected MUC15 expression majorly in placenta, thyroid gland, salivary gland and moderately in lungs and kidney. MUC15 protein is a 334 amino acid long polypeptide containing an amino terminal signal sequence, an extracellular tandem repeat domain, a small transmembrane region and a

2.1.1.11. Mucin 20. Mucin 20 is a membrane bound mucin encoded by MUC20 gene which is located on human chromosome 3q29. MUC20 102

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2008). They act as a physical barrier and protect the epithelium that lines the respiratory and gastrointestinal tracts by forming a matrix where bacteria are trapped. The gel forming mucins give a lubricant property to the mucus. Detailed structure of all secreted gel forming mucins are explained below. 2.1.2.1.1. Mucin 2. Mucin 2 was the first human secreted gel forming mucin to be identified and characterized and was initially cloned from a human intestine cDNA library (Gum et al., 1994; Gum et al., 1989). Mucin 2 was found to be expressed in small intestine (Audie et al., 1993; Buisine, 1999; Gum et al., 1994), colorectum (Audie et al., 1993; Gum et al., 1994) and airways (Copin et al., 2001; Buisine et al., 2000). MUC2 gene is located on human chromosome locus 11p15.5 (Desseyn et al., 1998; Griffiths et al., 1990; Rousseau et al., 2004). The mucin 2 monomer consists of more than 5000 amino acids. The central region consists of two tandem repeat regions. The first region is rich in serine/threonine/proline having 21 tandem repeats showing an irregular amino acid motif and the second region has 51–115 regular tandem repeats of 23 amino acid residues. Both tandem repeats are extensively O-glycosylated. This region constitutes more than half the length of mucin 2 protein. Cysteine-enriched domain (CYS domain) is present on either side of the first tandem repeat region. This domain is rich in cysteine and is not heavily glycosylated. The central tandem repeat domain is flanked on either side by domains as seen in Von Willebrand factor. Disulfide rich D-like domains namely, D1, D2, D’ and D3 domains are present in the N-terminal and D4, B, C and CK are present in the C-terminal (Fig. 3). They have role in oligomerization and dimerization, thus forming a highly viscous gel forming mucin network (Godl et al., 2002; Gum et al., 1994; Lidell et al., 2003a). D4 shows sequence homology to the D4 dimerization domain of vWF. A GDPH autocatalytic site is present near the C-terminal of mucin 2 (Lidell et al., 2003b). The cysteine knot domain is conserved with that of Von Willebrand factor and TGF-β and it mediates dimerization of mucin molecules. Studies have shown that mucin 2 has an important role in suppressing inflammation in the intestinal tract and thereby inhibiting the development of intestinal tumours (van der Sluis et al., 2006; Velcich et al., 2002). 2.1.2.1.2. Mucin 5AC. Mucin 5AC is a secreted gel forming mucin encoded by the gene MUC5AC that resides in the chromosome locus 11p15.5 (Pigny et al., 1996). It has a molecular weight of approximately 641 kDa. MUC5AC was initially cloned from a human tracheobronchial cDNA library. It is the major airway mucin produced by the goblet cells lining the airway (Buisine, 1999). In-situ hybridization and Northern blot analysis have shown that MUC5AC is most strongly expressed in submucosal glands and bronchial epithelium, surface mucous cells of gastric epithelium (Porchet et al., 1995), and conjunctival goblet cells of the eye (Gipson and Inatomi, 1997), moderately expressed in endocervix and weakly expressed in gallbladder (Porchet et al., 1995). It is a polymeric mucin made of 5525 amino acids. It consists of an amino terminal region, a central region and a carboxy terminal region. Mucin 5AC has heavily glycosylated mucin-like domains rich in proline, threonine and serine (PTS) residues interrupted by poorly glycosylated cysteine rich domains. The Nterminal region consists of cysteine rich domains D1, D2, D’ and D3 which have sequence similarity with the Von Willebrand factor (vWF) and a putative leucine zipper motif. The cysteine rich domains have role in disulphide-mediated polymer formation. The cysteine rich domain is made of 130 amino acids with 10 conserved cysteine residues (Guyonnet-Duperat et al., 1995; van de Bovenkamp et al., 1998). The central region is encoded by a single large exon containing nine cysteine domains of which cysteine1 to cysteine5 are interspersed by PTS rich domains without any repetitive sequences. Cysteine5 to Cysteine9 domains are interspersed by four tandem repeat domains of 8 amino acid residues (Guyonnet-Duperat et al., 1995). The most frequent consensus repetitive sequence is TTSTTSAP which contains numerous O-glycosylation sites. The carboxy terminal region consists of cysteine rich vWF-like domains D4, B, C and cysteine knot (Fig. 3). The

expression has been detected in kidney, oesophagus, stomach, duodenum and colon (Higuchi et al., 2004). The cytoplasmic tail of MUC20 is 503 amino acids long and has two functional domains in which one domain is involved in MUC20 oligomerization and the second domain has role in MUC20-Met binding. It consists of three 19amino acid tandem repeats with extensive O-glycosylation (Higuchi et al., 2004). Mucin 20 has two isoforms that differ in amino terminal sequence. 2.1.1.12. Mucin 21. MUC21 or epiglycanin belongs to the group of transmembrane mucins located on human chromosome locus 6p21.33. Studies have detected mucin 21 expression in lungs, thymus, large intestine and male reproductive organ. Mucin 21 glycoprotein is a 535 amino acids long polypeptide consisting of an N-terminal signal sequence, 28 tandem repeats of 15 amino acid residues rich in serine and threonine, stem domain with 22 amino acids, 23 amino acids long transmembrane domain, and a cytoplasmic tail of 64 amino acids (Fig. 2). Each tandem repeats contain 239 putative O-glycosylation sites. The mucin domain is poorly conserved while the non-mucin domains are highly conserved (Itoh et al., 2008). 2.1.1.13. Mucin 22. MUC22 is a cell surface associated mucin located on human chromosome 6p21.33. It is also known as panbronchiolitisrelated mucin-like 1 (PBMUCL1). MUC22 polypeptide is made of 1773 amino acids consisting of an amino terminal signal sequence, a mucinlike domain containing 124 tandem repeats rich in serine and threonine and each repeat is made of 10 amino acids and a carboxy terminal transmembrane domain (Fig. 2). The serine and threonine residues are O-glycosylated and MUC22 has 7 N-glycosylation sites. Studies have detected its expression in lungs, placenta and testis (Hijikata et al., 2011). 2.1.2. Secreted mucins Secreted mucins, produced by the goblet cells are characterized by their high molecular weight (5–40 MDa) and size (600–900 nm), high proportion of glycosylation (50–80%) and the capacity to form viscoelastic gels. The secreted mucins have been further subdivided into gel-forming mucins (MUC2, MUC5AC, MUC5B, MUC6, and MUC19) and non-gel-forming mucins (MUC7, MUC8 and OVGP1 or MUC9). 2.1.2.1. Secreted gel-forming mucins. Mucins synthesized by the goblet cells are the classical gel- forming mucins. It includes MUC2, MUC5AC, MUC5B, MUC6 and MUC19 which are secreted by intestine, stomach surface, salivary glands, stomach glands etc. In addition to the large central glycosylated PTS domain, these mucins are characterized by lightly glycosylated carboxy and amino terminal regions rich in cysteine. The cysteine enriched region (CYS Domain) is composed of N-terminal TIL domains (Trypsin inhibitor-like) and C8 domains (“C8” domain has eight conserved cysteines). Other domains include N- and/ or C-terminal Von Willebrand factor type D and C domains (vWD/vWC) which possesses sequence similarity to Von Willebrand factor and a Cterminal cysteine knot (CK) domain (Lang et al., 2007; Perez-Vilar and Hill, 1999; Zhou et al., 2012). All these domains have role in dimerization in endoplasmic reticulum through disulphide bond formation and their subsequent polymerization in trans-Golgi network to form multimers (Asker et al., 1998; Sheehan et al., 1996; Sheehan et al., 2004). MUC6 and MUC19 mucins also have the same domain structure, but lack the C-terminal VWD-C8-TIL unit. Mucins of the types MUC2 or MUC5 contain an additional domain known as CysD domain with a conserved WxxW motif that is present within the PTS region (Lang et al., 2007). MUC 2, MUC5AC, MUC5B and MUC6 are encoded by a cluster of genes located within 500 kb on the short arm of chromosome 11(11p15) (Pigny et al., 1996). The property of secreted gel forming mucins to oligomerize provide them the potential to build up dense visco-elastic mucus gel that covers many epithelia (Godl et al., 2002; Perez-Vilar and Hill, 1999; Sheehan et al., 1999; Thornton et al., 103

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Fig. 3. Structural diversity of different secreted mucins. The figure illustrates the domain structures of some secreted gel-forming and non-gel-forming mucins. The tandem repeats are interspersed by PTS domain (rich in proline, threonine and serine). D domains, Von Willebrand Factor type D domains (D1, D2, D’, D3, D4); B-like domain, Von Willebrand Factor type B domain; C-like domain, Von Willebrand Factor type C domain; CYS, Cysteine-enriched domain; CK, Cysteine Knot; GDpH, Gly-Asp-Pro-His; A3uD4, domain located between A3 and D4 domains in vWF.

gastric cDNA library (Toribara et al., 1993). Studies have detected the expression of mucin 6 in mucous neck cells of fundus, submucosal glands in antrum and cardia and Brunner’s glands of duodenum (Ho et al., 1995b). Weak expression has been detected in right colon and terminal ileum (Toribara et al., 1993; Toribara et al., 1997). MUC6 expression has also been detected in pancreas, gallbladder, seminal vesicles, female reproductive tract (Toribara et al., 1997; Gipson et al., 1997). Similar to other secreted gel forming mucins, Mucin6 also has a central glycosylated domain flanked by cysteine rich amino and carboxy terminal domains. The central tandem repeat domain has a 169amino acids consensus sequence rich in proline, serine and threonine that is heavily decorated with carbohydrate moieties. The N-terminal region consists of D1, D2, D’ and D3 domains that have similarity with Von Willebrand factor (vWF). The carboxy terminal domain contains two distinct regions in which one domain has high serine, threonine and proline content that is similar to the tandem repeat domain in amino acid composition and the second region is rich in cysteine residues (Fig. 3) and is almost 25% similar to the cysteine knot domain in Von Willebrand factor and human mucins located to the chromosome 11p15 such as MUC2, MUC5AC and MUC5B (Rousseau et al., 2004). In the gastric mucosa both MUC6 and MUC5AC act as a selective barrier for HCl diffusion. The O-glycans present in the tandem repeat domain of mucin 6 protects the gastric epithelium from H.pylori infection by inhibiting the biosynthesis of a major cell wall component, cholesteryl-alpha-D-glucopyranoside (Kawakubo et al., 2004). 2.1.2.1.5. Mucin 19. Mucin19 is a secreted gel forming mucin encoded by MUC19 gene, which is located on human chromosome locus 12q12. Mucin 19 has been detected in submandibular gland, sublingual gland, respiratory tract, eye and middle ear epithelium. MUC19 is translated into a polypeptide of 8384 amino acid residues. Its contains three amino terminal vWF D domains, followed by mucin-like tandem repeats rich in serine/threonine, a vWF C domain and a Cterminal cysteine knot domain (Fig. 3). Mucin 19 have role in ocular mucus homeostasis (Chen et al., 2004).

cysteine knot domain mediates dimerization by an autocatalytic process (Lesuffleur et al., 1995). A GDPH (Gly-Asp-Pro-His) autocatalytic proteolytic cleavage site is also seen towards the C- terminus. This site is demonstrated for MUC2, conserved in MUC5AC and MUC4 that cleaves between GD and PH residues. Mucin 5AC is the major mucus component in the normal respiratory tract epithelium that has an important role in defence against pathogenic and environmental challenges. Mucin 5AC in the gastric mucosa acts as a selective barrier for HCl diffusion. The glycan structures on MUC5AC, Leb and sialyl Lex act as ligands for Helicobacter pylori to adhere on gastric epithelium. Thus, MUC5AC has an important role in protecting the gastric epithelium from Helicobacter pylori infection. 2.1.2.1.3. Mucin 5B. Mucin 5B is a secreted gel forming mucin encoded by MUC5B gene that is located in the chromosome locus 11p15.5. It is a major human airway mucin produced in the mucous cells of submucosal gland and glandular ducts (Buisine, 1999). It is also known as high-molecular weight salivary mucin MG1 or human gallbladder mucin. Studies have detected MUC5B expression in bronchus glands (Audie et al., 1993), endocervix (Audie et al., 1995), gall bladder (Campion et al., 1995) and pancreas (Balague et al., 1994). MUC5B is the only human mucin gene that is not polymorphic in nature (Debailleul et al., 1998). MUC5B has a very large central exon encoding a peptide of 3570 amino acids that forms 19 subdomains. Most of these subdomains are similar to each other, thus forming four super repeat units of 528 amino acid residues. Each super-repeat is composed of 11 repeats of an irregular repeat of 29 amino acids, a unique sequence of 111 amino acid residues rich in serine, threonine and alanine and seven cysteine-rich regions made of 108 amino acids with 10 cysteine residues (Desseyn et al., 1997a). The carboxy terminal contains six subdomains made of 808 amino acids. It includes MUC11p15-type domain, a 56 amino acid domain similar to the A3uD4 domain (the domain located between A3 and D4 domains in vWF), D4-like domain, B-like domain made of 40 amino acids, C-like domain, and CK domain of 86 amino acid residues (Desseyn et al., 1997b) (Fig. 3). 2.1.2.1.4. Mucin 6. Mucin 6, the human gastric mucin is a secreted gel forming mucin encoded by MUC6 gene located on the human chromosome 11 in the region p15.5. MUC6 was initially cloned from

2.1.2.2. Secreted non-gel-forming mucins. MUC7, MUC8 and MUC9 are the non-gel-forming mucins. They cannot oligomerize since they lack 104

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like histatin and statherin that provide protection to the mucosal environment from invading pathogens and foreign particles by activating the immune system. All mucins contain proline, threonine and serine (PTS) rich repetitive domains. Serine and threonine are the sites for Oglycosylation and these glycosylated regions form the major binding site for many bacterial adhesins for stimulating immune response. Mucins also have direct antimicrobial activity against pathogens. It does not kill pathogens or inhaled contaminants, but trap them by forming viscoelastic gel and are cleared through mucociliary transport. Many clinical studies have revealed that mucin production increased not only in case of pathogen entry, but also due to inhaled substances (Basbaum, 1999). Mucins mainly restricted to the epithelial cell surfaces become exposed to circulation during pathogenic conditions and their over expression can be recognized as a potential biomarker for disease diagnosis (Chauhan et al., 2006). Different types of mucins are present throughout the body in specific locations. They have important role in maintaining homeostasis and promoting cell survival in different conditions. In the oral cavity, increased incidence of candidiasis and dental caries are caused by reduced levels of salivary mucins, which highlights the importance of salivary mucin in host defence and disease prevention. Salivary mucins such as Mucin5B (MUC5B) and Mucin7 (MUC7) can interact with oral microbes to facilitate their removal and reduce their pathogenicity with the help of antibacterial salivary protein (Frenkel and Ribbeck, 2015). The soluble mucin MUC7 has potent antimicrobial and antifungal activity in the salivary gland (Bobek and Situ, 2003). Gastric mucus is primarily composed of MUC5A and MUC6, which form a protective layer that act as a selective barrier for HCl and maintains a pH gradient between surface epithelium and the gastric lumen. It also plays an important role in pepsinogen transport and its activation inhibition. In addition, the adherent mucus acts as a barrier for luminal pepsin and thereby protecting the mucosa from proteolytic digestion (Bafna et al., 2008). Small intestinal mucus is constantly secreted from crypt mixes which contain antibacterial peptides and lysosome from paneth cell. Gel-forming mucin MUC2 is the major component of mucus in the small and large intestine. Large intestine has a different type of mucus organization than small intestine by having a two layered mucus system. The inner layer of mucus is formed from surface goblet cells. This inner layer is continuously renewed and protects the host epithelium from commensal bacteria. Its properties were also found to be coupled with immune system (Pelaseyed et al., 2014). Ocular mucins are produced by conjunctial goblet cells and play role in lubrication and ocular defence. Dry eye syndrome, where eye does not produce enough tears or tears evaporate too quickly, could be caused by reduced levels of mucin production in ocular region. These finding highlighted the importance of regulated mucin production in ocular surface (Corfield et al., 1997). For the treatment of dry eye syndrome, mucin can be used as a high molecular additive to improve the adherence of artificial tear drops (Bansil and Turner, 2006).

the cysteine rich domains and are thus found as monomers (Dekker et al., 2002; Levine et al., 1987). Different non-gel-forming mucins are discussed below. 2.1.2.2.1. Mucin 7. Mucin 7 belongs to the class of secreted nongel-forming mucins located on the chromosome locus 4q13.3 (Bobek et al., 1996). MUC7 was initially cloned from a human submandibular gland cDNA library (Bobek et al., 1993). It is synthesized by the serous cells of salivary gland. Mucin 7 expression has been detected in submandibular, sublingual and labial salivary glands. MUC7, also known as low molecular weight human salivary mucin (MG2), has a molecular weight of 120–150 kDa with the core protein being 39 kDa. The human salivary mucin, MUC7 is translated as a single polypeptide of 357 amino acids. The N-terminal consists of 144 amino acids of which the first 20 amino acid residues make up the leader peptide. The amino terminal has a histatin-like domain containing a leucine- zipper segment, two cysteine residues at positions 65 and 70, four potential Nglycosylation sites and nine O-glycosylation sites. Since MUC7 exist as a monomer, the two cysteine amino acids may be involved in intramolecular rather than inter molecular disulphide bond formation. The central region is made of 138 amino acid residues. It has six tandem repeat regions of 23 amino acids rich in glycosylated proline (35%), threonine (22%) and serine (17%). The tandem repeat region is flanked on either side by unique amino acid sequences, potential glycosylation sites and cysteine rich regions. Carboxy terminal is rich in proline (5 residues). It contains a leucine-zipper segment, one N-glycosylation site and 26 potential O-glycosylation sites (Bobek et al., 1993; Gururaja et al., 1998) (Fig. 3). MUC7 is an antimicrobial protein that have role in aiding the clearance of bacteria from oral cavity and also helps in mastication, speech and swallowing. 2.1.2.2.2. Mucin 8. Mucin8 is a secreted non-gel-forming mucin located on human chromosome locus 12q24.33 (Shankar et al., 1997). Studies have detected MUC8 expression in submucosal glands of the trachea, testis, placenta, cervix and endometrium with weak to undetectable levels in seminal vesicle, epididymis, fallopian tube, ovary and uterus (D’Cruz et al., 1996). It is a major airway mucin and a 313 amino acid partial polypeptide consisting of two types of consensus repeats and is rich in proline, serine, threonine, alanine and glycine (Shankar et al., 1994). 2.1.2.2.3. Oviductal glycoprotein 1/MUC9. Mucin9 is a secreted nongel-forming mucin located on the human chromosome locus 1p13.2 (Lapensee et al., 1997). It is expressed only in the female reproductive tract. MUC9 encodes oviductin protein which is an oviduct specific glycoprotein (Arias et al., 1994; Lapensee et al., 1997). It is a 678 amino acids polypeptide containing four N-glycosylation sites, two Nmyristoylation sites, and several phosphorylation sites (Arias et al., 1994). It may have role in protecting fallopian tube and early embryo (Lapensee et al., 1997). 3. Physiological functions of mucins

4. Mucin biosynthesis

Mucins execute wide range of physiological functions like protection and lubrication of epithelial surfaces, maintaining epithelial integrity, aiding in cell adhesion and confer protection against pathogens, toxins and foreign particles. Mucins has protective role against pathogenic organisms either directly or indirectly. It acts as an adhesive agent or forms a physical dynamic barrier. Usually, secreted mucins form a protective layer over epithelial lined surfaces of the respiratory, genitourinary, gastrointestinal and oculo-vestibular systems. The membrane anchored mucins function as cell surface receptors and respond to various external stimuli for different cell responses like cell growth, differentiation, cell proliferation and apoptosis. In addition to the protective barrier formation, complex mucin gels capture and hold the biologically active compounds such as trefoil factors, defensins and secreted immunoglobulins, thereby promoting wound healing and mucosal restoration at epithelial cell damaged sites (Hollingsworth and Swanson, 2004). It also holds antimicrobial agents

The translational product of secretory mucin gene (MUC2, MUC5AC, and likely MUC5B and MUC6) is cotranslationally synthesized and then quickly dimerized inside the endoplasmic reticulum (ER) of goblet cells using their C-terminal Cysteine knot (CK) domain (Asker et al., 1998). The CK domain contains about 215 cysteine residues. Before or after the immediate formation of dimer, N-glycosylation may take place. So, N-glycosylation is not required for the dimerization process. But in case of rat Muc2 and Muc5AC it has been reported that N-glycosylation is required for the dimerization process. In order to exit the MUC2 protein from ER, all cysteine residues need to be oxidized. Recently it has been found that one chaperone ERN2 (endoplasmic reticulum to nucleus signaling 2) (Bertolotti et al., 2001; Martino et al., 2013; Tsuru et al., 2013) and small PDI (Protein disulfide isomerase)like protein AGR2 (Anterior gradient protein 2 homolog) are involved 105

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(Lidell and Hansson, 2006; Lidell et al., 2003b; Soto et al., 2006). For secretory mucins, pH of ER is the main factor that determines the cleavage. The cleavage of MUC2 usually happens below pH 6, whereas MUC5AC cleavage occurs at neutral pH (Lidell et al., 2003b). In another study of mucin secretion by human epithelium in respiratory system, Li et al. found that myristylated alanine rich C kinase substrate (MARCKS) is the key molecule which regulates the secretion of mucin from mucin filled vescicles (Li et al., 2001b). Major intestinal as well as stomach mucins are mainly produced by specialized goblet cells. Mucus organization of large intestine and small intestine is quite different. The mucus layer of large intestine is divided into an inner layer and an outer layer. The fast turnover rate of intestinal mucus increases the secretion of MUC2 mucin by goblet cells (Fig. 5). During secretion, it gets unfolded and then interact with previously secreted mucus and finally forms a continuous network.

in the proper processing of MUC2 protein in ER. Usually C- terminal KDEL (Lys-Asp-Glu-Leu) sequence will help to localize the soluble proteins in the ER. In case of AGR2, a variant but unique ER localization C- terminal motif KTEL (Lys-Thr-Glu-Leu) is present, hence confined in ER (Higa et al., 2011; Gupta et al., 2011; Tsuru et al., 2013). It has been predicted that AGR2 sequesters MUC2 mucin covalently. Based on the study report of Park and his colleagues, it has been found that mice lacking AGR2 are more prone to colitis (Tsuru et al., 2013). But little is known about the role of AGR2 in the biosynthesis of MUC2. After the formation of dimer in ER, it apparently moves to the Golgi apparatus (Asker et al., 1998). Golgi is the major site of O-glycosylation. O-glycosylation begins when it reaches the cis-golgi compartments where Nacetylgalactosaminyl transferase, an enzyme confined to cis- golgi, forms the GalNAc(N-acetylgalactosamine)-Ser/Thr linkages in the two central PTS domain (Roth et al., 1994; Deschuyteneer et al., 1988). Subsequently, elongation of oligosaccharides continues in medial and trans-golgi compartments with the help of another enzyme known as glycosyltransferases (Van Den Steen et al., 1998). In trans-golgi, interchain disulfide bonds were formed through N-terminal D domains and finally organized into concatenated rings. Under slightly acidic pH and high concentration of calcium, mucins aggregates and are then packed into granules (Fig. 4A). So, the maintenance of pH is crucial for mucin aggregate formation (Ambort et al., 2012). The trans-Golgi achieves this slight pH with the help of bicarbonate and cystic fibrosis transmembrane conductance regulator (CFTR) channel (Gustafsson et al., 2012). Both secretory as well as membrane bound mucin genes are translated into a single polypeptide. During post translational processing of membrane bound mucins (MUC1, MUC3, MUC16, and MUC17), one unidentified protease cleaves the single polypeptide into two subunits. Cleavage mainly occurs at their evolutionary conserved SEA domain (Macao et al., 2006; Wreschner et al., 2002). After cleavage, the two subunits remain non-covalently bound together throughout their synthesis and finally gets transported to the cell surface (Fig. 4B). But in case of secretory mucins (MUC2, MUC4, MUC5AC) the story is different, because they lack a conserved SEA domain. Such types of mucins are cleaved at other specific sequence called GDpH (Gly-Asp-Pro-His)

5. Role of mucins in various pathological conditions Mucus are mainly composed of water, salt, proteins such as defensins, lysozymes, immunoglobulins, growth factors, trefoil factor, lipids, resistin like molecule β (RELMβ), FCγ binding protein (fcgbp) and mucins. Among these, mucins are the main component that gives viscous property to mucus. The mucins prevent the entry of pathogens and other harmful substances to the epithelium. The altered expression of mucins has been found in various diseases like cancers, inflammatory bowel diseases, ocular surface diseases etc. An outline of different mucins up regulated or down regulated in various pathological conditions is summarised in Table 2. 5.1. Expression of mucins in cancer Mucins have seen to be upregulated in different types of cancer. Hence currently used as a potential marker for disease diagnosis and also recognized as therapeutic targets (Rhodes, 1999). The hypersecretion of mucins favours cancer cells in several ways. Due to their characteristic pattern of glycosylation, it serves as a binding platform for various growth factors and cytokines and thereby promotes

Fig. 4. A. Schematic representation showing the biosynthesis of secretory mucins. The translational product of secretory mucin gene is quickly dimerized inside the ER via C-terminal CK domains. The N-glycoslated polypeptide chain further moves to the Golgi complex, where tandem repeats undergo O-glycosylation. Once the dimerized polypeptide reaches the transgolgi compartment, dimers link together to form multimers via N-terminal D domains. Finally, mucins are packed into vesicles and then fusion of these vesicles to the plasma membrane causes the release of secreted mucins outside. B. Schematic representation showing the biosynthesis of transmembrane mucins. After post translational modifications, the translational products of transmembrane mucin genes are finally delivered into apical side of the membrane.

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Fig. 5. Schematic representation showing the organization of mucus in large intestine.

haematological malignancies including lymphomas, multiple myeloma and myeloid leukemia. In chronic myelogenous leukemia blasts, MUC1 stabilizes BCR-ABL and thereby encourages proliferation but inhibits differentiation and apoptosis (Kawano et al., 2007). To date only the role of MUC1 in haematological malignancies is reported. Increased

proliferation and metastasis of cancerous cells through several signalling cascades (Singh et al., 2006). MUC1 transmembrane mucins suppress the inflammatory responses developed during the entry of pathogenic bacteria. Recently it has been reported that MUC1 mucins are aberrantly overexpressed in various

Table 2 Mucins in various pathogenesis. Disease

Type

Mucins

Up regulation/Down regulation

Reference

Cancer

Lung cancer

MUC1 MUC4 MUC1 MUC3 MUC5B MUC1 MUC4 MUC1 MUC3 MUC13 MUC11 MUC12 MUC2 MUC3 MUC1 MUC6 MUC5AC MUC1 MUC4 MUC13 MUC16

Up regulated Up regulated Up regulated Up regulated Up regulated Up regulated Up regulated Up regulated Up regulated Up regulated Down regulated Down regulated Up regulated Up regulated Down regulated Down regulated Down regulated Up regulated Up regulated Up regulated Up regulated

(Guddo et al., 1998; Hanaoka et al., 2001; Tsutsumida et al., 2007)

Breast cancer

Pancreatic Cancer Colorectal cancer

Gastric Cancer

Ovarian Cancer

Inflammatory bowel diseases

Ulcerative colitis Crohns disease

MUC2 MUC3 MUC4 MUC5B

Down Down Down Down

regulated regulated regulated regulated

Ocular surface diseases

Atopic keratoconjunctivitis Vernal keratoconjunctivitis Dry eye syndrome

MUC5AC MUC5AC MUC5AC

Down regulated Up regulated Down regulated

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(Apostolopoulos and McKenzie, 1994; Hayes et al., 1985; Kufe, 2008; Rakha et al., 2005) (Andrianifahanana et al., 2001; Balague et al., 1994; Choudhury et al., 2000a; Singh et al., 2007a) (Duncan et al., 2007; Walsh et al., 2007; Williams et al., 1999)

(Reis et al., 1997; Reis et al., 1999; Utsunomiya et al., 1998; Wang and Fang, 2003)

(Chauhan et al., 2006; Fritsche and Bast, 1998; Singh et al., 2008a)

(van der Sluis et al., 2006) (Buisine et al., 1999)

(Dogru et al., 2005) (Burgel et al., 2001) (Zhao et al., 2001)

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disease outcome, combined evaluation of mucins, MUC1 and MUC2 are clinically useful (Utsunomiya et al., 1998). In more than 90% of the gastric adenocarcinomas, the mucins carbohydrates including Tn, Sialyl-Tn and T antigens were involved (David et al., 1992). But the role of these antigens in the progression of cancer is still unclear. Expression of Tn is restricted in columnar cells while sialyl-Tn is predominantly seen in goblet cells (Carneiro et al., 1994). Sialyl-Tn is a marker of small intestinal mucosa and also a powerful indicator of gastric cancer progression (Iwata et al., 1993). The expression of MUC5AC is widely seen to be expressed in lower grade carcinoma compared to late stage (Reis et al., 1997; Reis et al., 1999). Increased expression of MUC5AC is associated with better prognosis while reduced expression indicates poor patient survival (Kocer et al., 2004). Approximately 64.9% gastric cancer patients and 91% intestinal type gastric cancer showed increased expression of MUC13. In 9 out of 10 cases of intestinal metaplasia (intestinal type gastric cancer), the expression of MUC13 has been detected.

level of MUC1N subunit in the serum can be used as a marker for breast cancer and its aberrant localization on non-apical side of membranes and cytosol indicates worse prognosis (Rahn et al., 2001). Nuclear localization of MUC1 has also been found in human breast carcinoma. Aberrant localization of MUC1 in mitochondria is associated with diminished apoptosis mediated DNA damage and apoptosis activating factors hence promote proliferation. MUC13 transmembrane mucins are reported to be overexpressed in ovarian (Chauhan et al., 2009a) and human colorectal carcinoma (Walsh et al., 2007). The aberrant expression of MUC1 is also found in various solid tumours. MUC16 has been reported to be over expressed in 80% cases of epithelial ovarian cancer. Over expression of MUC4 has been used as a potential biomarker for the diagnosis of pancreatic carcinogenicity (Jhala et al., 2006). The aberrant localization of MUC4 on the nonapical portion of the membrane has been found in various carcinomas including pancreas, breast, lungs, ovary and gallbladder. Recently it has been demonstrated that during prostate (Singh et al., 2006) and urothelial cancer (Kaur et al., 2014), the expression of MUC4 markedly decreases. This report clearly indicates the complicated context dependent property of mucins. Specific inactivation of MUC2 gene in mice induce tumor in colon, small intestine and rectum. These results typically indicate the role of MUC2 in tumor suppression. However, the exact role of MUC2 in tumor suppression still remains unclear.

5.1.3. Colorectal cancer The increased expression of tumour associated glycoprotein (TAG72) has been observed in 45% of colorectal carcinoma patients (Okudaira et al., 2010). A study was conducted in 462 colorectal carcinoma samples for evaluating the expression of MUC1 and MUC3. Their findings suggest that about 74% of tumour samples showed the expression of MUC3 whereas 32% showed the expression of MUC1 (Duncan et al., 2007) (Fig. 6). Those patients who expressed MUC1 showed poor survival. Increased expression of MUC13 has been observed in poorly differentiated colon tumor (Walsh et al., 2007). Recently it has been identified that the major colon mucins MUC11 and MUC12 were down regulated during colorectal carcinogenesis (Williams et al., 1999) (Fig. 6). During adenocarcinoma there is a different pattern of sulphation and acetylation in mucins. Adenocarcinoma mucins showed a significant reduction in O-acetylation and sulphation, but a significant increase in sialylated mucins (Kim et al., 1996). Compared to adenocarcinoma the phenotype of mucinous carcinoma is different. In mucinous carcinoma condition, there is hypersecretion of mucus linked with changes in O-acetylation and sialic acid content (Hanski et al., 1997). The reduced chain length in mucins’ oligosaccharide has also been found in colorectal cancers (Kim et al., 1996). Especially the expression of truncated version of sialyl-Tn, Tn and T has been reported in 90% of colorectal cancer (Springer, 1997; Steven et al., 1989). Fetal gut expresses specific normal blood group antigens including Lea, A, B and Leb blood group antigens but immediately shut down the expression after birth. But in colorectal cancers these blood group antigens are aberrantly re-expressed along with some other incompatible blood group antigens.

5.1.1. Breast cancer Differential expression of both membrane bound (MUC4, MUC1 and MUC16) as well as secreted mucins (MUC5AC, MUC5B, MUC6 and MUC2) have been widely observed in breast cancer (BC) tissues compared to normal breast tissues. The study in breast cancer model suggests that the BC development is mainly through a chain of events and is believed to progress from ductal hyperplasia/typical epithelial hyperplasia, atypical ductal hyperplasia, carcinoma in situ and invasive adenocarcinoma. Studies showed that the gene expression as well as copy number of MUC1 is remarkably high in breast cancer cells, hence it can be used as a potential target for immunotherapy and diagnostic assays (Apostolopoulos and McKenzie, 1994). The expression of MUC5B widely seems to be upregulated in sclerosing papillomas and fibroadenomas (Fig. 6). The differential expression of MUC1, MUC5AC, MUC5B, MUC6 and MUC2 have been observed in ductal carcinoma in situ, while in lobular carcinoma in situ the MUC2 expression is upregulated. In BC tissue models, membrane bound mucins such as MUC3, MUC1 and MUC4 showed 91%, 77% and 95% positivity respectively while secreted mucins such as MUC2, MUC5AC, MUC5B and MUC6 showed 19%, 37%, 19% and 100% positivity respectively. Notably the modulation of expression of secreted mucins has been observed compared to normal breast epithelium. Mucinous carcinomas are another rare pathological condition accounted for an estimated 2% cases of BC. Furthermore, numerous experimental evidences demonstrated an increased level of MUC1-N subunit (CA15.3) in the serum of BC patients (Hayes et al., 1985; Kufe, 2008). Also found its aberrant localization in cytosol as well as non-apical side of the membrane indicating worse prognosis (Rahn et al., 2001). The expression of MUC3 is also found to be overexpressed in 91% of breast cancer tissues (Fig. 6). The aberrant expression of MUC3 in BC tissue membrane confers worse prognosis, higher Nottingham Prognostic Index (NPI) and tumor grade (Rakha et al., 2005).

5.1.4. Lung cancer Expression of MUC1 and MUC4 is found to be overexpressed in squamous cell cancer of lung (Guddo et al., 1998) (Fig. 6). Increased expression of both mucins showed poor patient survival and prognosis (Hanaoka et al., 2001; Tsutsumida et al., 2007). 5.1.5. Ovarian cancer In majority of advanced ovarian cancer, the expression of MUC16 (CA125) found to increase (Chauhan et al., 2009b) (Fig. 6). Hence it can be used as a potential prognostic as well diagnostic marker (Fritsche and Bast, 1998; Singh et al., 2008a). Recently it has been found that the expression of MUC4 and MUC1 is remarkably elevated in ovarian cancer, suggesting the combined assessment of all these mucins to increase the sensitivity of late stage tumour diagnosis (Chauhan et al., 2006) (Fig. 6). In addition, immunohistochemical analysis of epithelial ovarian cancer samples showed a significant increase in the expression of MUC13 compared to normal tissues. The highest expression of MUC13 has also been observed in mucinous epithelial ovarian cancer

5.1.2. Gastric cancer A study was conducted in 46 gastric carcinoma tumour samples for evaluating the expression of MUC2, MUC1, MUC3, MUC6 and MUC5AC. The expression of MUC1, MUC5AC and MUC6 were observed in normal gastric samples. In metaplasia condition, the expression of MUC2 and MUC3 were increased but the expression of MUC1, MUC5AC and MUC6 decreased (Fig. 6). The key finding of this study was the increased expression of MUC1 associated with better prognosis (Wang and Fang, 2003). It has been suggested that in order to predict the 108

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Fig. 6. An outline of the upregulation and downregulation of mucins in different cancers.

(Chauhan et al., 2009a, 2009b) (Fig. 6). MUC4 is a yet another best studied mucin in ovarian cancer which is overexpressed in 100% cases of early stage ovarian cancer. Hence combined evaluation of MUC16 and MUC4 may be useful for achieving higher sensitivity for the assessment of late stage tumors.

1A to PanIN-1B, PanIN-2 and PanIN-3. The expression of MUC1 and MUC4 is found to be higher as it progresses to higher grade PanINs. The expression of other mucin genes such as MUC11 and MUC12 has been reported in pancreatic cancer. However, their significance in relation to pancreatic cancer has not been fully uncovered.

5.1.6. Pancreatic cancer MUC6, one of the best studied mucin is usually expressed on normal pancreatic tissue. MUC6 and MUC1 are expressed on the fetal pancreas at early stage of gestation, while the expression of MUC1 and MUC5AC on adult pancreas seems to be low (Ohuchida et al., 2006). Normally the expression of MUC1 is confined on the apical portion of intralobular ductules of normal pancreas, while its expression is entirely absent in larger ducts and islet of langerhans. However, the level of expression of MUC1 is significantly altered during the early stages of pancreatic cancer, which further increases with the progression of invasive carcinoma (Balague et al., 1995) (Fig. 6). The elevated level of mucin associated antigens such as CA50, CA19.9 and CA195 were found in pancreatic cancer (Bhargava et al., 1989; Magnani et al., 1983; McLaughlin et al., 1999). The expression of MUC4 is completely absent in normal pancreas or chronic pancreatitis. Studies have shown that the expression of MUC4 is highly elevated in pancreatic tumour cell lines and human pancreatic tumors (Andrianifahanana et al., 2001; Balague et al., 1994; Choudhury et al., 2000a) (Fig. 6). Thus, MUC4 have found to be one of the potent biomarker for the diagnosis of pancreatic cancer (Andrianifahanana et al., 2001; Singh et al., 2007a). Pancreatic cancer development model study suggests that it develops through a continuum of stages starting from a lesion known as pancreatic intraepithelial neoplasia (PanIN), to flat without atypia, papillary without atypia, papillary with atypia and finally develops into lesions with severe atypia. Lesions with severe atypia are further classified into PanIN-

5.2. Expression of mucins in bowel disease MUC2 is one of the major secretory mucin that is expressed in the outer and inner layer of intestinal mucus. MUC2 protects the intestinal epithelium from pathogenic bacteria and also from other hazardous environmental factors that are exposed in lumen. Usually the outer layer of the intestinal mucosa is less dense and therefore MUC2 in this layer is more susceptible to proteolytic cleavage by commensal bacteria. But the inner layer is densely packed and less exposed and hence the bacterial colonization is completely absent (Johansson et al., 2008). Any alteration in this microenvironment will enhance inflammatory responses at the underlying epithelial layer. 5.2.1. Ulcerative colitis Ulcerative colitis, a multifactorial inflammatory bowel disorder is linked with a reduction in goblet cells in colorectal mucosa (Fig. 7). Hence the production of mucin is reduced, thereby reducing the thickness of mucus (Pullan et al., 1994). Ulcerative colitis patient’s intestinal mucosa exhibits an increased expression of sialomucins and a reduced expression of O-acetyl sialic acids and sulphomucins. Depletion of O-acetylation is an indication of severity of ulcerative colitis. Chronic inflammation increases the risk of colon cancer. Based on recent data, it has been found that the mice lacking both MUC2 and an anti-inflammatory cytokine interleukin10 (IL-10) are more susceptible to ulcerative colitis (van der Sluis et al., 109

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Fig. 7. Schematic representation showing the reduction of goblet cells in colorectal mucosa during ulcerative colitis.

patients with sjogrens syndrome. But in case of OCP patients, the cell layer and cell type distribution of conjunctival polypeptide-galNActrasferases is altered. This altered increase in polypeptide-galNActransferases is altered in the apical cells of ocular surface is characterized by drying and reduced keratinization.

2008). Still it is not known whether other than MUC2 any other mucins are involved in the protective role of the intestinal mucosa. 5.2.2. Crohns disease Crohns disease is a yet another multifactorial inflammatory-bowl disease that affects any part of gastrointestinal tract. These patients have heavily O-acetylated mucin in the termini of ileum. Based on the comparative analysis of healthy and crohns disease histological data, it has been found that the crohns disease patient’s ileum showed a reduced expression of the MUC3 and MUC4 and in intestinal mucosa, MUC5B expression is also reduced compared to normal (Buisine et al., 1999).

5.4. Regulation of MUCINS in various pathological conditions 5.4.1. Role of cytokines in mucin regulation Cytokines are potent immune cell products which play diverse role in various pathophysiological processes. There are mainly two types of primary inflammatory cytokines: The T helper 1(TH1 or type 1) and T helper 2(TH2 or type 2). Type 1 cytokines (IL-2, TNF, IFNγ and IL-12) enhance cellular immune responses and Type 2 cytokines (IL-6, IL-4, IL5, IL-9, IL-13 and IL-10) favours humoral immune responses (Lucey et al., 1996; Seder and Paul, 1994). Although, evidences suggest that in a variety of cell types cytokines specifically induce mucin transcription by activating Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway (Boudny and Kovarik, 2002; Pfitzner et al., 2004; Shuai and Liu, 2003). Type 1 cytokines have been shown to upregulate the expression of membrane bound mucins. While type 2 cytokines were found to be associated with the upregulation of secreted mucins. MUC1 induction by interferon γ (IFNγ) has been reported in ovarian and breast cancer cell lines (Clark et al., 1994; Gaemers et al., 2001). In another instance, IFNγ regulated upregulation of MUC4 has been reported in pancreatic tumor cell lines. More recently, Tumor Necrosis Factor α (TNF α) of type 1 cytokine has been shown to upregulate the transcription of MUC1 in nasal epithelial cells (Shirasaki et al., 2003). It has been speculated that this event might contributes to upper airway system inflammatory diseases (Fahy, 2002). Moreover, the cytokines IFN γ and TNF have shown to induce MUC1 synergistically in human normal as well as malignant breast cancer cell lines (Lagow and Carson, 2002). The hypersecretion of secreted mucin by type 2 cytokines has been widely studied in airway inflammatory diseases. Although among type 2 cytokines, the role of IL-4, IL-9 and IL-13 has been extensively studied. Usually IL-4 activates STAT-6 through shared IL-4 receptor (Chomarat and Banchereau, 1998; Takeda, 1996). The selective expression of IL-4 in transgenic mice induced the expression of MUC5AC in non-ciliated airway epithelial cells, but it did not induce MUC2 expression (Temann et al., 1997). Intranasal installation with IL-13 in BALB/c showed goblet cell metaplasia along with MUC5AC gene upregulation in lung tissues (Zuhdi et al., 2000). Also, constitutive expression of IL-13 showed an enhanced expression of MUC5AC gene in airway epithelium of transgenic mice models (Zhu et al., 2001; Zhu et al., 1999). IL-9 yet another type 2 cytokine, has been shown to elevate the expression of MUC2 and MUC5AC in airway epithelial cells of transgenic mice which strongly suggests its role as a key regulator of asthmatic response (Kim, 1997). However, it is not

5.2.3. Ileoanal pouch A notable change can be seen in the patterning of mucin sulphation and O-acetylation in ileum and colon within six to nine months of pouch formation (Corfield et al., 1992). Usually the level of sulphation and O-acetylation is low in normal ileum and in newly formed pouch mucosa. But in established pouch mucosa the level of sulphation and Oacetylation shows a 72% increase compared to normal (Shepherd et al., 1993). 5.3. Ocular surface disease 5.3.1. Ocular allergies Atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) are the most common ocular surface allergic disease linked with alterations in secreted and cell surface associated mucins. AKC patients are characterized by significant reduction in goblet cells and squamous metaplasia of conjunctival epithelium. In AKC patients due to the loss of goblet cells, the level of expression of MUC5AC is drastically decreased. This leads to loss of lubrication followed by epithelial cell damage. In order to compensate the loss of mucin 5AC, the level of expression of MUC1, MUC2 and MUC4 is increased (Dogru et al., 2006; Dogru et al., 2005). VKC patients are mainly characterized by inflammation and infiltration of eosinophils in ocular surface (Bonini et al., 2004). In VKC patients the number of goblet cells in conjunctiva is high, hence the level of expression of goblet cell specific MUC5AC is increased (Aragona et al., 1996). Activated eosinophils are thought to be the major reason for upregulation of MUC5AC (Burgel et al., 2001). 5.3.2. Dry eye Sjogrens syndrome and ocular cicatrical pemphigoid (OCP) are systemic autoimmune diseases characterized by alterations in ocular surface mucins. Patients with Sjogrens syndrome have a decreased expression of conjunctival epithelium and decreased levels of goblet cell mucin MUC5AC in conjunctival epithelium and tears (Zhao et al., 2001). Decreased expression of MUC19 has also been reported in 110

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block the proliferation and metastasis of malignant cells. Aberrant localization of MUC1C in nucleus helps it to interact directly with the DNA binding domain of ERα (Estrogen receptor α), which further promotes the growth of the malignant cells (Fig. 8) (Wei et al., 2006). Roy et al. found that aberrant expression of MUC1 initiates Epithelial Mesenchymal Transition (EMT) in cancer cells (Roy et al., 2011). Further studies showed that the association between androgen receptor and MUC1C is critical for EMT in prostate cancer cells. Additionally, the direct interaction of MUC1C with the DNA binding domain of androgen receptor suppresses the expression of androgen receptor and this in turn leads to aggressive phenotypical changes in prostate cancer cells (Rajabi et al., 2012). In pancreatic cancer cells, the aberrant overexpression of MUC1 is associated with an increase in the expression of EMT related transcription factors such as SLUG and SNAIL. Further the studies in mice model suggests that knock down of Muc1 significantly reduce the Epithelial Mesenchymal Transition (Roy et al., 2011). Aberrant pattern of glycosylation and palmitoylation in MUC1 have also been observed in malignant cells. Recently reported that the aspartic acid at 36th position of MUC1C is aberrantly modified by N-glycosylation and acts as a docking site for galectin3. Galectin3 in turn helps MUC1 to interact with EGFR. This interaction further prevents the degradation of EGFR and promotes the constitutive EGFR signaling in cancer cells. Other experimental reports demonstrated that the inhibition of MUC1 attenuates growth of EGFR and Transforming Growth Factor alpha (TGFα) induced tumorigenesis. The recycling of MUC1 is associated with palmitoylation of CQC motif of MUC1C domain. During malignancy, CQC motif plays a pivotal role in the oligomerization of MUC1C. In cancerous cells, MUC1C is released from the cell membrane and aberrantly accumulates in the cytosol. In cytosol MUC1C undergoes oligomerization which is essential for targeting MUC1C to either nucleus or mitochondria. Once entered into mitochondria, it inhibits the release of apoptogenic factors. Emerging evidences suggests that inhibition of MUC1C oligomerization is one of the highly effective methods for killing MUC1 positive prostate and human breast cells (Raina et al., 2009; Joshi et al., 2009). In response to stress, FGF induces phosphorylation at cytoplasmic domain of MUC1 which facilitates its binding to heat shock protein 90 (HSP 90) and finally MUC1-HSP90 complex moves to mitochondria (Ren et al., 2006). This aberrant localization attenuates stress induced release of apoptogenic factors in mitochondria. Agata et al., showed that MUC1C attenuates the activation of extrinsic apoptotic pathway by direct interaction with FADD and hence blocks its recruitment to caspase8 (Agata et al., 2008) (Fig. 9). Usually normal epithelial cells utilize this mechanism for their protection. But malignant cells exploit this mechanism for its survival. The cytoplasmic domain of MUC1C has 12 putative phosphorylation sites and serve as docking site for various kinases including Protein Kinase C delta (PKCδ) (Ren et al., 2002), Zeta-chain-associated protein kinase 70 (ZAP70), Src family kinases (Li et al., 2004; Li et al., 2003; Li et al., 2001a), glycogen synthase kinase 3β (GSK3β) (Li et al., 1998), receptor tyrosine kinases and abelson murine leukemia viral oncogene homolog (ABL) (Raina et al., 2006). The MUC1C activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway through direct interaction with IκB, REL-associated protein (RELA) and Iκα. IκBα undergoes phosphorylation in presence of IκB kinase complex which further leads to the degradation of IκBα. Hence it constitutively activates the NF-κB pathway and inhibit apoptosis (Ahmad et al., 2007). During oxidative stress, MUC1C regulates p53 mediated transcription by directly binding to the regulatory domain of p53 and selectively transactivate the genes required for growth arrest and inhibits the transcription of genes required for apoptosis (Chao et al., 2000; Wei et al., 2005) (Fig. 9). In addition, MUC1 also activates the transcription of an anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL) (Raina et al., 2004) and a transcriptional factor Forkhead box O3(FOXO3a) (Yin et al., 2004). The activation of FOXO3a is mainly regulated by Akt/PKB pathway. The key protein Phosphoinositide 3-

clear whether IL-13 is directly involved in gene activation of mucin or it is simply one of the intermediate of pathways. More recently, the role of EGFR mediated IL-13 induced expression of MUC5AC in experimental animal models has been reported. 5.4.2. Regulation of transmembrane mucins in cancer The extra cellular domains of membrane bound mucins are homologous to epidermal growth factor (EGF) family. The exact function of this juxta membrane domains is not clear, but it has been postulated that this region interacts with the EGF receptor family and function in differentiation, proliferation, growth and inflammation related signalling cascades. Certain mucins such as MUC3A, MUC3B, MUC12, MUC13, MUC4 and MUC17 have 2–3 EGF domains but for MUC3A, MUC12, MUC17 and MUC13 mucins the SEA regions separate EGF domains. The extracellular juxtamembrane domain of MUC4 shows a sequence similarity with heregulins and hence it can interact with human epidermal growth factor receptor (HER2/neu/ERBB2) (Hollingsworth and Swanson, 2004). The sequence of EGF domain of MUC12 shows a significant similarity with several growth factors like Milk fat globule-EGF factor 8 protein (MFG-E8), crypto-1, Epidermal growth factor (EGF), heparin binding EGF, amphiregulin, β-cellulin and TNF- α (Transforming Growth Factor α). It has been speculated that the release of extracellular domain may expose the EGF-like motif so that it can interact with EGF receptor (ERBB) family or with other associated receptor for performing its functions such as growth, differentiation and proliferation. Tumour cells exploit this role of transmembrane mucins for performing their functions. Cancerous cell changes the glycosylic pattern of mucins and then aberrantly express them on its membrane. The role of MUC1 and MUC4 in carcinogenesis is highly elucidated. 5.4.2.1. MUC1. Aberrant expression of MUC1 is found to be associated with about 80% of human cancers. Recently it has been reported that the cytoplasmic domain of MUC1 (MUC1C) has several phosphorylation sites, which interact with various growth factor receptors and other signaling molecules inorder to inhibit apoptosis, block death receptor and also regulate the transcription of several genes for promoting proliferation, finally converting into a malignant phenotype (Singh and Hollingsworth, 2006). Schroeder JA. et al., showed that over expression of MUC1 induces a spontaneous mammary gland tumor in laboratory mice model, suggesting its pivotal role in malignancy (Schroeder et al., 2004). However, available evidences showed that the cytoplasmic domain of MUC1 regulates proliferation via ERBB dependent and independent mechanism. It was found that upon activation of MUC1C, it interacts with ErbB receptor and activates ERK1 and ERK2 (Extracellular Signal Regulated Kinases) thereby enhancing the proliferation (Schroeder et al., 2001). In case of Erb-independent mechanism, MUC1C activates ERK signaling via MEK1, Raf-1 and cjun (Hattrup and Gendler, 2006). In addition, MUC1 also interacts with other receptors such as PDGFRβ (Platelet-derived growth factor receptor beta) (Singh et al., 2007b), Met (Singh et al., 2008b) and fibroblast growth factor receptor 3 (FGFR3) (Ren et al., 2006). The stimulation of Fibroblast growth factors (FGF) in breast cancer cells induces phosphorylation on YEKV (Tyr-Glu-Lys-Val) motif of MUC1C that enhances the binding affinity of β-catenin (Wnt pathway). Finally, MUCIC-β-catenin complex gets localized in the nucleus (Ren et al., 2006). Here β-catenin acts as a co-activator of cell-cycle progression genes cyclin-D1 and c-myc. The members of src family (c-Src, Lyn and Lck) has also been shown to induce phosphorylation on YEKV motif of the MUC1C domain. This aberrant phosphorylation in-turn acts as a docking site for β-catenin and (Fig. 8) promotes the proliferation and survival of malignant cells (Schroeder et al., 2003). The association between β-catenin and MUC1C was identified in various cancers including human gastric (Udhayakumar et al., 2007), pancreatic (Singh et al., 2007b; Wen et al., 2003), colorectal (Baldus et al., 2004) and breast (Schroeder et al., 2004). Another experimental report suggests that the attenuation of both FGF and ErbB are sufficient to 111

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Fig. 8. Schematic diagram representing the different mechanisms of MUC1 for cell proliferation: Cytoplasmic domain (CT) of MUC1 interacts with β-catenin and further increases its level in cytoplasm and nucleus by inhibiting GSK3β mediated degradation of β-catenin. Further β-catenin contributes to cell proliferation by increasing the expression of cell cycle progressing genes such as cyclin D and c-myc. Src family members such as Lyn, Lck and c-Src also increases cell proliferation by interacing with the CT of MUC1. MUC1 CT also regulates proliferation by stabilizing ER-α.

of which the β subunit participates in intracellular signaling. MUC4 regulates cellular growth mainly through ERBB2 dependent and ERBB2 independent pathway. MUC4 acts as an intramembrane ligand of ERBB2/HER2 receptor and facilitates the dimerization of the receptors (Carraway et al., 2002; Singh et al., 2004). This dimerization induces cross-phosphorylation at specific tyrosine residues on cytoplasmic domain of MUC4 (Schlessinger, 2000). The aberrant expression of MUC4 in malignant condition induces constitutive signaling of ERBB2, which further promotes cell proliferation and disrupts cell-matrix and cell-cell interactions (Komatsu et al., 1997) by regulating several signaling molecules such as FAK, P3IK and MAPK (Hollingsworth and Swanson, 2004). MUC4 induced relocalization of ERBB2 from lateral to apical region has been observed in polarized colon cancer cells CACO-2. After the relocalization of ERBB2, MUC4 serves as a ligand for ERBB2. Upon ligand binding, the dimerized receptor induces cross-phosphorylation at 1139Y and 1248Ysites (Ramsauer et al., 2006). This phosphorylation activates AKT through p38 Mitogen-activated protein kinase (MAPK). Horowitz et al. showed that p38 mediated activation of AKT promotes the survival of cells (Horowitz et al., 2004). It has been reported that aberrant overexpression of MUC4 confers apoptotic resistance to tumor cells in laboratory rat. Emerging evidences suggest that not only MUC4 and ERBB2 interaction is involved in anti-apoptotic mechanism but also several other signaling pathways imparts in this mechanism. Therefore, further studies are required to decipher the exact signaling pathways. Recently Rachagani et al., identified the involvement of MUC4 in initiating epithelial mesenchymal transition in pancreatic cancer cells

kinase (PI3K) phosphorylates and deactivates FOXO3a and it is retained in cytoplasm. Upon activation, FOXO3a immediately moves into the nucleus and activates the expression of certain set of genes which are thought to be responsible for scavenging the reactive oxygen species (ROS) formed during oxidative stress (Fig. 9). In vitro studies in cancerous cells showed that MUC1 inhibits PI3K-Akt/PKB pathway while its attenuation inactivates FOXO3a. However, the overexpression of MUC1 and its subsequent cleavage and altered localization facilitate cancer cells to escape from apoptotic mechanisms, emphasizing the importance of MUC1 in malignancy. Toll-like receptors (TLRs) are membrane receptors which recognize a unique pattern in microbes known as pathogen-associated molecular patterns (PAMPs) (Janssens and Beyaert, 2003; Takeda and Akira, 2005). After this recognition step, TLRs elicit immune response against the invading pathogenic microbes. Recently it has been reported that the expression of TLRs is high in almost all cancers, indicating its immense role in cancer progression. Experimental evidences demonstrated that the cytoplasmic domain of MUC1 modulates TLR signaling. But how MUC1 regulates this TLR mediated signaling is still unknown. Recently it has been speculated that the recognition of aberrantly expressed TLRs by altered glycosylated MUCs is thought to be responsible for the aberrant activation of various cytokines and chemokines such as G-CSF, TNFα, IL-6, IL-1, IL-8, MCP-1 and COX-2 thereby making that environment more suitable for cancer progression (Huang et al., 2008). 5.4.2.2. MUC4. During synthesis MUC4 undergoes autocleavage at specific GDPH (Gly-Asp-Pro-His) site to generate 2 subunits α and β 112

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Fig. 9. MUC1 signaling pathway for survival and suppression of apoptosis. MUC1 specifically transactivates FOXA3a, p53 and NF-κB, and suppress apoptosis. Activated MUC1C forms a complex with HSP70 and HSP90, which then transduces signal to mitochondria, where it attenuates the apoptotic pathway. MUC1 also forms complex with the death effector domain FADD, where it inhibits the activation of extrinsic apoptotic pathway.

et al., 2003; Rakha et al., 2005; Wang and Fang, 2003). Mass ARRAY analysis in CpG sites upstream of MUC3A distal promoter sites in lung, pancreas, breast and colon cancer cells revealed the correlation between methylation and expression of MUC3A. Hypomethylation causes decreased expression of MUC3A while hypermethylation restores its expression (Kitamoto et al., 2010). But histone H3 modifications do not have any role in regulation of MUC3A. Detailed evaluation of epigenetic modification of MUC4 in cancer cells showed that methylation at CpG sites in 5′ flanking region and histone H3 modification at k9, k27 regulate its expression (Vincent et al., 2008). Recent studies in pancreatic and pancreatic ductal carcinoma tissue samples showed the aberrant expression of MUC4 mRNA, suggesting the aberrant hypomethylation at CpG sites (Zhu et al., 2011).

(Rachagani et al., 2012). Moreover, experimental evidences demonstrated that MUC4 overexpressed cancer cells have been associated with an increase in the expression of EMT related transcription factors such as TWIST, ZEB1 and SNAIL (Ponnusamy et al., 2008). MUC4 suppression in pancreatic cancer induces a change in the phenotype of cells, reduced the expression of mesenchymal cell specific marker such as N-cadherin and Vimentin and upregulates the expression of epithelial specific markers such as cytokeratin-18, Ecadherin and occludin (Rachagani et al., 2012). 5.4.3. Epigenetic regulation Studies related with epigenetic regulation of mucin genes are seen to be low. But emerging evidences suggests the significant role of epigenetic mechanism in regulation of mucin gene. Recently Yamada et al. found that gene expression of MUC1 is mainly regulated by DNA methylation in CpG islands upstream of promoter region and histone H3 modification at lysine 9 near start site (Yamada et al., 2008). The epigenetic role of MUC2 gene expression is widely studied compared to MUC1. Studies in lung, pancreas, colon and breast cancer cell lines showed that the MUC2 gene expression is mainly regulated by CpG methylation, histone H3-K4 methylation, histone H3-K9/K27 acetylation and histone H3-K9 dimethylation (Hamada et al., 2005; Yamada et al., 2006; Yamada et al., 2010). Studies showed that hypomethylation of MUC2 gene is one of the major reason for the overexpression of MUC2 mucin in mucinous colon carcinoma cells (Okudaira et al., 2010). The aberrant expression of MUC3A has been found in various cancers including pancreatic, gastric, renal and breast. But the functional role of MUC3A in the development of cancer is not yet clear. However, it was found that the expression of MUC3A in malignant condition is associated with poor prognosis (Leroy et al., 2003; Park

5.4.4. Mucin regulation by bacterial products Mucins are one of the major barrier which prevents the entry of many bacteria. Some of the pathogenic strains of bacteria compete with this protective mechanism of mucus and final successfully adhere to the underlying epithelial cells. This includes Helicobacter pylori, Campylobacter jejuni, Citrobacter rodentium, Salmonella enteric, Listeria monocytogenes, parasite Entamoeba histolytica, Porphyromonasgingivalis, E. coli, Pseudomonas, Streptococcus and Pneumococcus. Among these, Helocobacter pylori and Citrobacter rodentium successfully cross the mucin barrier and then effectively colonize beneath the inner layer. Listera monocytogenes utilize goblet cells and mucins for their colonization. Normally E-Cadherin is hidden inside the goblet cells and it will be exposed only during the emptification of Goblet cells. During the entry of this pathogenic strain, Goblet cells start to secrete more mucins for preventing its colonization. This results in the emptification of Goblet cells and there by exposing the hidden E-cadherin. Subsequently 113

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Fig. 10. Schematic representation showing the mechanism of Helicobacter pylori infection and its related host immune response: One of the bacterial enzyme urease neutralizes the acidic pH in the stomach which facilitates its colonization in gastric epithelium. Adhesins mediated bacterial attachment in the gastric epithelium favours the transfer of VacA and CagA inside the host cell, which will induce strong inflammatory responses in gastric mucosa. Lipopolysaccharide (LPS) of the H.pylori activates specific Toll like Receptors (TLR4 and TLR2), which further leads to the activation of a transcription factor NF-κB. The activated NF-κB induces the expression of a variety of inflammatory molecules by regulating a series of signalling pathways which ultimately attract immune cells towards infection site to produce cytokines.

1996). More recently, other findings have established lipoteichoic acid from Staphylococcus aureus as a potent mucin stimulator in human NCIH292 and HM3 cells. Furthermore, lipothecoic acid activates ADAM-10 (A disintegrin and metalloprotease) dependent cleavage and then trigerring the release of membrane proHB-EGF through G-protein-coupled platelet-activating factor receptor. This released HB-EGF (Heparin binding EGF) subsequently activates Ras/Raf/MEK/ERK/pp90/NF-KB pathway and finally induces the activation of MUC2. Helicobacter pylori and Porphyromonas gingivalis derived LPS exhibited an inhibitory effect on mucin expression and promoted apoptosis in primary cultures of rat gastric mucosal cells and sublingual salivary gland of acinar cells respectively.

the bacteria interact with E-cadherin and successfully colonize inside the stomach. The parasite Entamoeba histolytica and Porphyromonasgingivalis secrete proteases that specifically cleave the MUC2 (Lidell et al., 2006; Van der Post et al., 2013). Bacterial infection is one of the common cause of inflammation in airway, middle ear and digestive tract. Reid suggested that the respiratory epithelium continuously produces a particular amount of mucin at baseline and during the exposure to any pathogenic bacteria or other environmental contaminants, epithelial cells increase the production of mucin (Reid, 1963). LPS (Lipopolysaccharides) present on the cell wall of gram negative bacteria induce inflammation as well as mucin gene expression through various complex mechanisms. Lipid A moiety of LPS molecule represents a major stimulator for mucin gene activation. Studies have shown that transtympanic injection of LPS in rats induced mucous cell hyperplasia in middle ear. Several other in vitro experimental studies have established a significant correlation between LPS and mucin gene expression. Pseudomonas aeruginosa, a gram negative pathogenic strain of bacteria usually colonizes the lungs of cystic fibrosis patients. It induces the transcription of MUC2 mucin by activating NF-κB through MAP kinase signaling pathway (Li et al., 1997). Moreover, Pseudomonas aeruginosa derived LPS mediated upregulation of MUC5AC has been established in human HT29-MTX and NCI-H292 cell lines. Recently, the involvement of Src-dependent ERK pathway has been identified in LPS-mediated MUC2 regulation in human HM3 and NCI-H292 cancer cells. But Staphylococcus aureus, a gram-positive bacterium induces the transcription of MUC2 and MUC5AC through platelet activating factor receptor (Cundell et al.,

5.4.4.1. H.pylori associated mucin regulation and associated inflammation. Helicobacter pylori infection increases the risk of gastric cancer. Approximately 75% of gastric cancer cases are associated with Helicobacter pylori infection. Therefore in 1994 the International Agency for Research on Cancer classified H.pylori as type 1 carcinogen (Working Group on the Evaluation of Carcinogenic Risks to Humans, 1994). During the entry, bacteria release urease enzyme to the gastric environment, which hydrolyzes urea into carbon dioxide and ammonia that neutralize the acidic pH (Hatakeyama, 2009) and also induces a reversible alteration in the glycosylation of mucins (Ota et al., 1998), which favours its attachment. Helicobacter pylori infected patients showed an alteration in the expression of MUC6 and MUC5AC. Histological analysis showed an increase in expression of MUC6 and decrease in expression of MUC5AC (Byrd et al., 1997). After the 114

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6.1.2. Tecemotide Tecemotide is (previously known as Stimuvax or L-BLP25) a liposome based investigational therapeutic cancer vaccine that induces immune response by targeting MUC1 antigen (Sangha and Butts, 2007). Tecemotide was developed by Canadian biotech company Biomira Inc. It is now under investigation for the treatment of different MUC1 over expressed cancers such as breast cancer, prostate, colorectal cancer and lung cancer. At present, Tecemotide in undergoing phase III clinical trials for non-small cell lung cancer. Both TG4010 as well as Tecemotide have no negative impact on health-related quality of life (Rotonda et al., 2015).

attachment, it secretes several proteases and sulphatases that causes the degradation of gastric mucus (Sidebotham et al., 1991; Slomiany and Slomiany, 1992). After crossing the mucus layer of stomach, the bacteria express two types of adhesions SabA (sialic acid binding adhesion) and BabA (blood group antigen binding adhesion) which favours its attachment to gastric epithelium. This attachment activates several other virulence factors such as CagA (Cytotoxin associated gene A antigen), VacA (vaculatingcytotoxin), IceA (induced by contact with epithelium), DupA (duodenal ulcer-promoting gene) and OipA (outer inflammatory protein). SabA and BabA mediated adhesion facilitates the transfer of CagA and VacA virulence factors into the host cell. These factors further activate several inflammatory pathways in the body which develop strong immune response in host body. This immune response in presence of H.pylori increases oxidative as well as genotoxic stress that leads to the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that favours oncogenic mechanisms. Along with this, Toll like Receptors mainly TLR4 and TLR2 recognize lipopolysaccharide (LPS) of H.pylori and activates transcription factor NF-κB. The activated NF-κB moves into nucleus and activates the expression of several cytokines and chemokines which are related to inflammation (Kumar pachathundikandi et al., 2011). It was found that NF-κB also activates other set of genes which are thought to be responsible for inflammation and cancer. This includes TRAF5, TRAF2, iNOs, RIP, IKKB and TAK1 genes. Furthermore, the activated cytokines recruit neutrophils, monocytes/macrophages, dendritic cells and lymphocytes which secrete various pro and anti-inflammatory cytokines leading to a chronic inflammation (Fig. 10).

6.1.3. PANVAC PANVAC is a cancer vaccine therapy regimen composed of two viral vectors such as recombinant vaccinia and recombinant fowl pox. Both vectors have transgenes for MUC1 specific TAAs. Now PANVAC is undergoing phase II clinical trials against colon and breast cancer. Clinical trials demonstrated its safety and ability to stimulate immune system in malignancies. Recent studies suggest that the combined therapy of docetaxel and PANVAC in metastatic breast cancer can provide a clinical benefit (Heery et al., 2015). 6.2. Antibody based therapy Transmembrane mucins which are over expressed in tumor conditions can be targeted for the anti-neoplastic vaccine development or monoclonal antibody generation. Mucin based antibody-drug conjugate is composed of a monoclonal antibody against mucin epitope coupled with an anticancer drug. This formulation can improve the side effects of chemotherapy because antibody-drug conjugates are more specific to tumor antigens. Antibody-drug conjugates developed against tumor antigen can trigger potent T-cell response and antibody dependent cell cytotoxicity. Additionally, monoclonal antibodies can elicit anticancer response at specific site by targeting mucin antigens overexpressed in tumors. Toxins or radionuclides conjugated anti-mucin antibodies have been tested as therapeutic treatments for several malignancies. MUC1 has been effectively used as a marker for disease diagnosis and as a target for immune-directed therapies. MUC1 specific antibodies targeting MUC1N subunit have not proved to be effective, because these antibodies need to be overcome the large pool of circulating MUC1N in order to reach the surface of cancer cells (Kufe et al., 1984). To avoid this problem, antibody against MUC1C domain has been developed. Many anti-MUC1 antibodies are now available against different cancers. For example, PankoMab, a humanized monoclonal antibody recognizing the tumor specific epitope of mucin-1 (TA-MUC1) developed by Glycotope, exhibits strong antibody dependent cell cytotoxicity. It has the ability to differentiate between tumor MUC1 and non-tumor MUC1 epitopes and is highly specific to the breast, gastric, colorectal, liver, cervical and thyroid tumors. Now, Pankomab is undergoing phase II trials for TA-MUC1 positive ovarian cancer. Oregovomab (OvaRex) has been developed as a potential therapeutic agent for patients with ovarian cancer. It is an anti-CA-125 antibody B43.13, which forms immune complexes with circulating CA125 and generates a broad cellular and humoral immune response (Berek et al., 2009). OvaRex is now in phase II clinical trial. Like MUC1, MUC16 is also overexpressed in ovarian cancer cells, which represents a potential target for therapeutic antibodies. An antibody conjugate with cytotoxic auristatins against the MUC16 tandem repeats has been found to be active against human OVCAR3 ovarian tumour xenografts in mice. Also, Chen et al. developed two monoclonal antibodies namely 11D10 and 3A5, and conjugated it to cytotoxic drug Mono Methyl Auristatin E (MMAE). Among these, 11D10 targeted nonrepeating epitope in the extracellular domains and 3A5 recognizes tandem repeats of MUC16 (Chen et al., 2007). 3A5 is more effective than 11D10 in promoting cell death by site specific delivery of cytotoxic drug to cancer cells. Presently, 3A5-MMAE conjugate referred to as

6. Mucin as therapeutic targets Currently we have sufficient information about mucins’ involvement in malignancies. It is known that the expression of mucin is significantly altered during different pathological conditions and tumorigenesis. Abnormality in glycosylation and overexpression of mucins in malignant cells are attractive targets for the development of novel therapeutic agents. These ideas provide a new platform for the development of safe and effective mucin based therapeutic agents. Many mucins based therapeutic agents (Table 3) are under different stages of clinical trial. Vaccines, antibodies, miRNAs, cell based therapy and drug inhibitors are the different types of therapeutic formulations which specifically targets mucin glycoprotein (Fig.11). 6.1. Vaccines Vaccine is a biological preparation that enhances immune response against diseases/tumors. Vaccination is the most effective method to activate cell mediated immunity or humoral immunity by enhancing dendritic cell mediated tumor antigen presentation. MUC1 is one of the most studied tumor associated antigens (TAA) abnormally expressed in several malignant conditions. MUC1 targeted vaccines that are under different stages of clinical trials include TG4010, Tecemotide and PANVAC. 6.1.1. TG4010 TG4010 is a modified vaccinia virus Ankara based therapeutic cancer vaccine which is designed to target MUC1 TAA and interleukin 2 of cancerous cells. It is an attractive target for cancer immunotherapy developed by Transgene SA. TG4010 combined with chemotherapy have been efficiently used against non-small cell lung cancer (Arriola and Ottensmeier, 2016). In addition to lung cancer, TG4010 targets MUC1 TAA expressed in breast, colorectal, kidney and prostate cancers. Its safety and activity have been evaluated in Phase II studies in several types of solid tumors. Its biomarker activity has been identified (Limacher and Quoix, 2012). 115

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Table 3 Mucins as possible therapeutic agents. Therapeutic agent Type

Name of therapeutic agent

Immunotarget

Disease

Drug inhibitors

GO-201 GO-202 GO-203

MUC1 MUC1 MUC1

PMIP Geldanamycin 17-(allyl amino)- 17-demethoxy geldanamycin

MUC1 MUC1 MUC1

Breast cancer & prostate cancer Breast cancer & prostate cancer Cutaneous T cell lymphoma Acute myeloid leukemia (AML) Breast cancer Multiple myeloma &AML Multiple myeloma &AML

Antibody based therapy

Pankomab Oregovomab DMUC5754A Mab-DT3C Mab-PAM4

MUC1 MUC16 MUC16 MUC13 MUC1

Colorectal & gastric cancer Ovarian cancer Ovarian cancer Pancreatic cancer Pancreatic cancer

Phase 2 Phase 2 Phase 1

(Berek et al., 2009) (Berek et al., 2009) (Felder et al., 2014) (Nishii et al., 2015) (Gold et al., 2007)

Vaccines

TG4010 Tecemotide (Stimuvax or L-BLP25)

MUC1 & IL-2 MUC1

MUC1

Phase Phase Phase Phase Phase Phase Phase

(Arriola and Ottensmeier, 2016) (Sangha and Butts, 2007)

PANVAC

Non-small cell lung cancer Non-small cell lung cancer Multiple myeloma Rectal Cancer Prostate cancer Pancreatic cancer Bladder, colorectal & breast cancer

miR145 miR1291 miR200c miR132 miR150

MUC13 MUC1 MUC4 MUC16 MUC13 MUC4

miR29a miR3305p miR21913p

MUC1 MUC1 MUC4

Pancreatic cancer Oesophageal cancer Pancreatic cancer Pancreatic cancer Pancreatic cancer Malignant melanoma & Pancreatic cancer Pancreatic cancer Pancreatic cancer Pancreatic cancer

CVac

MUC1

Ovarian cancer

miRNA based therapy

Cell based therapy

Clinical trial stage

Ref

(Joshi et al., 2009) (Raina et al., 2009) (Joshi et al., 2009) Phase 2 (Bitler et al., 2009) (Yin et al., 2010) (Yamada et al., 2010)

2 3 2 2 2 3 2

(Heery et al., 2015) (Sachdeva and Mo, 2010) (Luo et al., 2015) (Radhakrishnan et al., 2013) (Radhakrishnan et al., 2013) (Zhan et al., 2014) (Srivastava et al., 2011) (Trehoux et al., 2015) (Trehoux et al., 2015) (Jonckheere et al., 2015)

Phase 2

(Gray et al., 2016)

Fig. 11. Schematic representation showing different therapeutic agents used for mucin targeting. A. Antibody based therapy: Mucin extracellular domains targeted by anti mucin antibody conjugated with radionuclides or toxin. B. Drug inhibitors as therapeutic agent: drug inhibitors are small molecules or peptides that bind to c terminal domain of mucins and prevent its localization into mitochondria or nucleus. C. Cell based therapy: mucin epitope containing dendritic cell used for targeting mucin.

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homodimerization of MUC1. They have arginine residues at the N terminal for cell permeability and amino acid sequence of MUC1C CQC motif (Yin et al., 2010). GO-201, GO-202, GO-203, PMIP, Geldanamycin and 17-(allyl amino)- 17-demethoxy geldanamycin specifically target and inhibit MUC1C to control different types of human cancers. GO-201 and GO-202 are two small peptides developed by Genew Oncology that recognize MUC1C CQC motif responsible for the translocation of MUC1-C to various subcellular organelles. In vivo and in vitro studies showed that both of them have anti tumorgenic activity and prevent MUC1 localization into mitochondria or nucleus (Raina et al., 2009). GO-201 treatment is associated with inhibition of breast and prostate cancer cell growth in vitro and animal models (Joshi et al., 2009). GO-203, another peptide inhibitor of MUC1C in cutaneous T cell lymphoma (CTCL) cells, induced apoptosis mediated by reactive oxygen species (ROS) and also inhibited CTCL cell growth in murine xenograft models. PMIP is an inhibitory peptide used against MUC1C cytoplasmic domain. The cytoplasmic domain is the binding site for β-catenin as well as it acts as a potential substrate for EGFR and SRC phosphorylation (Bitler et al., 2009). Treatment with PMIP has been associated with the induction of human breast cancer cell death in in vitro as well as in vivo studies. Small molecular inhibitors can be used to target promoters of Rab and other trafficking proteins to reroute MUC to degradation pathways. Geldanamycin and 17-(allyl amino)- 17-demethoxy geldanamycin blocked the trafficking of MUC1 to mitochondria and also reduced FGF1 induced MUC1 interaction with HSP90 (Ren et al., 2006). Many studies shown that in vitro and in vivo studies of MUC1C inhibitors have induced ROS mediated cell death in both multiple myeloma and acute myeloid leukaemia cells (Stroopinsky et al., 2003; Yin et al., 2010). Thymoquinone, a natural alkaloid, down regulates the expression of MUC4 and hence it can be used for treating MUC4 mediated cancers (Torres et al., 2010).

DMUC5754A, is undergoing phase I clinical trial (Felder et al., 2014). The adhesion of ovarian cancer cells to the peritoneum is based on the interaction between MUC16 and mesothelin. An anti MUC16 antibody against binding domain on mesothelin can block the interaction between MUC16 and mesothelin. This is also a potential immunotherapeutic agent. For example, HN125 is an immunoadhesin which contains fused product of MUC16 binding epitope of mesothelin and Fc portion of human IgG1 antibody, which prevents the adhesion of ovarian cancer cells by blocking the interaction between mesothelin and MUC16 (Radhakrishnan et al., 2013). The sialyl Tn and sialyl T epitopes’ expression are very limited in normal tissues whereas it is highly expressed in tumor antigens of adenocarcinoma. These sites were specifically targeted using monoclonal antibodies such as CC49 and B72.3. At present, radioimmune conjugates of these two antibodies are undergoing clinical trials for pancreatic, breast and colorectal adenocarcinoma. Nishii et al. developed a MUC13 targeting monoclonal antibody designated as TCC56 (Nishii et al., 2015). This mAb conjugated with diphtheria toxin containing recombinant protein DT3C is found to be cytotoxic to pancreatic cancer cells. The mAb-DT3C conjugate promotes cancer cell death by targeting MUC13. Combined radio and immune therapy can increase the efficiency and tumor response. Mab-PAM4 is a promising antiMUC1 antibody which reacts with 85% of pancreatic adenocarcinoma. Gold et al. reported that low dose I131 -PAM4 antibody along with gemcitabine may provide an improved alternative for the treatment of pancreatic cancer (Gold et al., 2007). Furthermore, Posey et al. developed a MUC1Tn glycoform recognizing chimeric antigen receptors (CARs) (Posey et al., 2016). T cell leukemia and pancreatic cancer have been successfully controlled by antiTnMUC1 CAR T cells that inhibited the growth of tumors in xenograft models. These findings revealed the therapeutic benefits of CAR T cells and engineered T cells for immuno specific tumor therapy. 6.3. Drug inhibitors

6.4. miRNA based therapy Drug inhibitors are small peptides or molecules which specifically bind and inhibit oncogenic behaviour of target molecules. MUC1 is a well-studied mucin gene and several MUC1C drug inhibitors have been reported to control its oncogenic nature (Fig. 12). Oncogenic nature of MUC1 is due to homodimerization of MUC 1C through amino terminal CQC motif. Several peptide drug inhibitors were developed to block

miRNAs are a class of non-coding RNAs complementary to target sequence and post transcriptionally regulates gene expression. It is a novel potential therapy for controlling cancer gene expression through RNA silencing and acts as tumor suppressors (Gayral et al., 2014). At present several miRNAs are available to target different MUC genes.

Fig. 12. Schematic representation showing MUC1 as an attractive therapeutic target for different disease therapy: MUC1 targeting using different types of microRNAs and drug inhibitors.

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Sachdeva and Mo reported the significance of miR145 as a tumor suppressor by targeting MUC1 (Sachdeva and Mo, 2010). miR145 specifically inhibits MUC1 expression thereby reducing cell invasion and metastasis. Also, recent studies reported that targeting the oncogene cMyc through miR145 helps to suppress tumor cell growth both in vivo and in vitro (Sachdeva et al., 2009). Trehoux et al. identified two new tumor suppressive miRNAs such as miR29a and miR3305p that inhibit MUC1 expression (Trehoux et al., 2015). These two miRNAs have strong inhibitory effect on MUC1 expression by directly binding to MUC1 3′UTR (Un Translated Regions) in pancreatic cancer cells. miR1291 is used for targeting MUC1 to regulate growth, invasion and apoptosis of oesophageal cancers. It is a new therapeutic strategy for treating oesophageal squamous cell carcinoma (Macao et al., 2006). Over expression of MUC13 is one of the major reasons for the development of pancreatic tumour. Zhan et al. identified two tumor suppressing miRNAs namely miR-145 and miR-132 for pancreatic cancer therapy (Zhan et al., 2014). Among these, miR-145 has better inhibitory effects on MUC13 compared to miR-132 and thus it can be used as an important tool for improving cancer therapy. Jonckheere et al. reported miRNA mediated targeting of MUC4 in pancreatic cancer (Jonckheere et al., 2015). In pancreatic cancer, MUC4 aberrantly regulates various signalling pathways and hence its inhibition is important to suppress oncogenic behaviour. miR21913p is a tumor suppressor that inhibits MUC4 expression in pancreatic cancer (Jonckheere et al., 2015). MUC4 and MUC16 expression levels are seen to be upregulated in pancreatic cancer. miR200c has a potential inhibitory role in pancreatic cancer by regulating the expression of MUC4 and MUC16 (Radhakrishnan et al., 2013). Moreover, miR-150 is another tumor suppressor in pancreatic cancer cells and malignant melanoma. It down regulates the expression of MUC4 in both cells. In normal cells, MUC4 is regulated through transcriptional level mechanisms (Srivastava et al., 2011).

conserved SEA domain while in case of secretory mucin, cleavage occurs at GDPH sequence. Many studies have shown the altered/overexpression of mucins in various pathological conditions, including cancer. The hypersecretion of mucins in cancers is due to their characteristic pattern of glycosylation, which serves as a binding platform for various growth factors and cytokines and thereby promote the proliferation and metastasis of cancerous cells through several signalling cascades. Also, aberrant localization of mucins in mitochondria promotes proliferation through diminishing apoptosis activating factors. Each mucin has specific role in tumor induction; some of MUC genes shows hyper activation and others shows inactivation in pathological conditions. For example, the specific inactivation of MUC2 is found in many tumors and the aberrant expression of MUC1 is found in various solid tumors. On the basis of the increased level of mucin in the serum, it can be used as a potential marker for disease diagnosis and prognosis. Cytokines and transmembrane mucins are mainly involved in the regulation of mucus level in different pathological conditions. There is also significant correlation between bacterial products and mucin gene expression. Emerging evidences suggest the significant role of epigenetic mechanism in regulation of mucin gene expression. Our knowledge on protein architecture of mucins and its role in signalling and pathogenesis provide a new avenue for the therapeutic targeting of mucins. Many mucins based therapeutic agents are under different stages of clinical trial. Vaccines, antibodies, miRNAs, cell based therapy and drug inhibitors are the different types of therapeutic formulations which specifically targets mucin glycoprotein. We expect that majority of mucins would also be identified as direct drug targets with effective and safe therapeutic potential in the near future.

6.5. Cell based therapy

Suresh Sulekha Dhanisha, Sudarsanan Drishya and Prathapan Abeesh acknowledge University Grants Commission (India), Council of Scientific and Industrial Research (India) and Department of Biotechnology (India) respectively for providing financial support in the form of Junior Research Fellowship. The authors are thankful to Dr. Paul Sebastian, Director, RCC and Dr. S. Kannan, Head, Division of Cancer Research, Regional Cancer Centre (RCC) for providing valuable support required for the study.

Conflict of interest No conflict of interest. Acknowledgements

Cell based therapy is a novel treatment method for site specific mucin targeting. Immature dendritic cells isolated from patients will be incubated with mucin epitope encoding DNA. The mucin antigen containing activated dendritic cells are injected into the patient to specifically target MUC glycoprotein and stimulate immune system. Mucin derived peptides containing dendritic cells were able to induce a potent T- cell response and provide clinical benefit. CVac is a dendritic cell based vaccine that targets MUC-1 glycoprotein in epithelial ovarian cancer. It is a strong immuno stimulant/natural killer cell stimulant developed by Prima Biomed. CVac is safe and well accepted in ovarian cancer patients. It is currently undergoing phase 2 clinical trial (Gray et al., 2016).

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