Regulation of mucin 1 expression and its relationship with oral diseases

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(1)UEF//eRepository DSpace Rinnakkaistallenteet. https://erepo.uef.fi Terveystieteiden tiedekunta. 2020. Regulation of mucin 1 expression and its relationship with oral diseases Kashyap, Bina Elsevier BV Tieteelliset aikakauslehtiartikkelit © 2020 Elsevier Ltd. CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ http://dx.doi.org/10.1016/j.archoralbio.2020.104791 https://erepo.uef.fi/handle/123456789/8331 Downloaded from University of Eastern Finland's eRepository.

(2) Journal Pre-proof Regulation of mucin 1 expression and its relationship with oral diseases Bina Kashyap, Arja M. Kullaa. PII:. S0003-9969(20)30169-2. DOI:. https://doi.org/10.1016/j.archoralbio.2020.104791. Reference:. AOB 104791. To appear in:. Archives of Oral Biology. Received Date:. 30 January 2020. Revised Date:. 5 May 2020. Accepted Date:. 29 May 2020. Please cite this article as: Kashyap B, Kullaa AM, Regulation of mucin 1 expression and its relationship with oral diseases, Archives of Oral Biology (2020), doi: https://doi.org/10.1016/j.archoralbio.2020.104791. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier..

(3) Regulation of mucin 1 expression and its relationship with oral diseases. Running title: Mucin 1 in oral diseases and cancer. ro of. Bina Kashyap, Arja M. Kullaa*. -p. Institute of Dentistry, University of Eastern Finland, Kuopio campus, and Educational Dental Clinic, Kuopio University Hospital, Kuopio, Finland. Bina Kashyap. re. PhD Student, Institute of Dentistry, University of Eastern Finland, Kuopio campus, and Educational Dental Clinic, Kuopio University Hospital, Kuopio, Finland. lP. Email Id: Bina.kashyap@uef.fi. Arja M. Kullaa*. na. Professor. Department of Oral Diagnostic Sciences, Institute of Dentistry, Faculty of Health Sciences, University of Eastern Finland, Kuopio Campus, Finland. Jo. ur. Email: arja.kullaa@uef.fi. *. Corresponding Author:. Arja Kullaa, Professor Department of Oral Diagnostic Sciences, Institute of Dentistry, Faculty of Health Sciences, University of Eastern Finland, Kuopio Campus, PO Box 1627, FI-70211 Kuopio, Finland Email: arja.kullaa@uef.fi.

(4) Highlights . Transmembrane MUC1 is expressed on the apical surface of oral epithelial cell surfaces. Transmembrane MUC1 is a specific signalling receptor.. . MUC1 has an anti-inflammatory effect.. . MUC1 plays the role of tumor growth and metastasis.. re. -p. ro of. . Abstract. lP. Objective – The aim of this study is to describe the polymorphic mucin 1 (MUC1), and to provide an overview of the known complex and multiple functions of MUC1 in normal oral mucosa and oral mucosal lesions in compromised situations as well as exploring the. na. challenges associated with the heterogeneous nature of MUC1. We will review the current knowledge and provide insights into the future management possibilities of using MUC1 as a therapeutic agent.. ur. Methods – A literature search of the electronic databases included MEDLINE (1966 December 2019) and hand searches of cross-references were undertaken using terms related. Jo. to mucins, MUC1.. Results: MUC1 is a large transmembrane glycoprotein expressed on the apical surface of most of epithelial cell surfaces. Not only is it involved in lubrication, cell surface hydration, and protection against degrading enzymes, MUC1 also promotes abnormal cellular signalling, angiogenesis, anti-adhesion and tumorigenesis. Aberrant glycosylation, overexpression, loss of apical constraint are characteristics of the transformation of a normal cell to a cancerous cell. This review summarizes studies of MUC1 expression and function.

(5) with a special emphasis on oral epithelial cells in normal and abnormal conditions. In addition, current knowledge of MUC1 and unexplored areas of MUC1 are presented. Conclusion: MUC1 is an archetypical transmembrane protein, the presence of MUC1 in ectopic regions may lead to dysregulation of certain enzymes and activation of various pathways, favouring the development of inflammatory responses and tumour formation. This review examines the potential of MUC1 in the development of future therapeutics.. re. -p. ro of. Keywords: Epithelium, Mucin, Oral mucosa, Polymorphic, Tumorigenesis.. lP. 1. Introduction. The oral cavity is protected from foreign insults by its first line of defence - saliva. The composition of saliva has been extensively studied and it has been confirmed that the major. na. glycoprotein component present is mucin. From 1980 onwards, mucin has been a muchstudied feature in the research field due to two main observations: 1) the unexpected complex. ur. glycosylated structure of mucin as demonstrated by its structural chemistry and 2) the identification of the presence of tumor associated epitopes on mucin (Hanisch & Muller,. Jo. 2000).. Based on their biochemical and biophysiological characteristics, mucins can be categorized into three main categories: 1) Secreted/ Gel forming, 2) Membrane bound and 3) Soluble mucins (Moniaux, Escande, Porchet, Aubert & Batra, 2001; Kufe, 2009). A total of 21 mucins have been identified in humans to date, of which 12 are membrane bound (MUC1, 3A, 3B, 4, 12, 13, 14, 15, 16, 17, 21, 22) and 7 mucins are known to be secreted or soluble (MUC2, 5AC, 5B, 6, 7, 19 & 20). The domain architecture is unknown for MUC8 and MUC9 (Kumar et al, 2017). The main secretory and soluble mucins present in saliva are MUC 5B &.

(6) MUC 7 whereas MUC1 is the membrane bound mucin in the major and minor salivary glands as well as in oral epithelial cells. Secreted mucins and non-immune host defence components of saliva are swallowed shortly after secretion whereas after secretion, MUC1 resides on the cell surface and is involved in epithelial cell protection (Chang, Chang, Kim, Leeb & Kho, 2011). Saliva is constantly secreted and swallowed but the salivary proteins may be unevenly cleared depending on their location in the oral cavity (Amerongen, Bolscher & Veerman, 1995). MUC1 is a transmembrane glycoprotein/mucin, providing a mucosal barrier against pathogen invasion. It has a widespread tissue distribution. MUC1 consists of three domains: one large. ro of. extracellular domain and two small transmembrane and cytoplasmic domains. The extracellular domain of MUC1 makes it highly polymorphic and rigid in structure due to the variable number of tandem repeats of certain amino acids as well as its extensive propensity for glycosylation (Patton, Glender & Spicer, 1995). The intracellular cytoplasmic tail plays a. -p. role in cell signalling. Most mammalian species show a high degree of sequence conservation in the MUC1 cytoplasmic domain and hence, most studies have focussed on the functional. re. importance of the cytoplasmic domain (Pemberton, Rughetti, Taylor-Papadimitriou & Gendler, 1996).. lP. Several isoforms of MUC1 (MUC1/TM, MUC1/SEC, MUC1/Y, MUC1/X and MUC1/Z) have been isolated and studied intensively due to their role in cancer metastasis (Kumari & Sudandiradoss, 2013). The MUC1 isoforms demonstrate a diversity of properties and. na. functions leading to disturbances in many physiological and pathological processes if there is MUC1 over-expression. The expression of all isoforms is associated with the presence of malignancy except for MUC1/SEC, its expression can be detected in non-malignant tissue. ur. due to its lack of transmembrane and cytoplasmic domain. All mucins except MUC4 contain Sea Urchin sperm protein, Enterokinase and Agrin (SEA) domain in the extracellular region.. Jo. Soon after its synthesis, MUC1 undergoes proteolytic cleavage in its extracellular domain within the SEA module (Levitin et al, 2005). The cleavage generates two subunits α and β that bind together in a complex structure which remains fixed to the cell surface. The function of MUC1 relies on an autoproteolytic event within the cytoplasmic domain; after cleavage, it displays a functional heterodimeric structure (Kumari & Sudandiradoss, 2013). The protective effect of MUC1 is important to defend oral epithelial surfaces from various noxious, pathogenic and non-pathogenic microbes at least to some extent. If there are.

(7) changes in the expression of MUC1 by oral mucosal epithelial cells, this is indicative that some alteration has occurred in the salivary composition and mucosal defence; this can be important in defence against oral mucosal infections, oral mucosal diseases and oral cancers. The aim of this paper is to systematically review the complex role of MUC1, to evaluate its expression, and the potential effect of changes in the expression level in salivary composition, external and internal environment. We hope to provide the basic knowledge to make it possible to understand the unique actions of MUC1 with respect to oral epithelial cells and oral lesions. We also provide a basis for further studies which can focus on oral cancers and immunotherapy targeting MUC1.. ro of. 2. Search strategy. The Online English literature search and PubMed databases screened by two investigators to retrieve relevant studies published before 31 Dec 2019. The search criteria used various. related key words such as “MUC1”, “Mucins”, “MUC1 and Microplicae”, “Microplicae”,. -p. “normal oral mucosa mucins”, “epithelial dysplasia”, “oral cancers”, “oral squamous cell carcinoma”, “MUC1 expression”, and “MUC1 gene expression”; in order to widen our. re. search, references from the articles were manually examined, to check if they had not been identified during the database search. Titles and abstracts were screened for subject. lP. relevance. Those articles relevant to the oral mucosa and oral lesions were included in the study. Even articles, which mentioned an area of research with a minor emphasis on oral mucosa or any correlation with oral mucosa, were also included if they had information on. na. the structure, function and mechanism of MUC1 expression. 3. MUC1 Function. ur. There is a carbohydrate-enriched membrane bound biological coating called glycocalyx, attached to the cell membrane and it is within this environment that mucins perform their. Jo. numerous functions. The high degree of O-glycosylation of mucin MUC1, along with MUC5B and MUC7 prevents dehydration of the oral mucosa through continuous lubrication, stabilization of mucous gel at cell surface as well as exerting an antibacterial effect and conferring protection from proteolysis (Brayman, Thathiah & Carson, 2004; Nath & Mukherjee, 2014). MUC5B (high molecular weight oligomeric mucin) and MUC7 (low molecular weight monomeric or dimeric mucin) are the two major mucins in saliva (Takehara, Yanagishita, Podyma-Inoue & Kawaguchi, 2013). MUC5B is the primary gel forming mucin whereas MUC7 lacks gel-forming properties. MUC7 has the ability to self-.

(8) associate to form a highly ordered assembly which is important in promoting bacterial aggregation. MUC5B protects the oral cavity by binding to antibacterial salivary proteins (Frenkel & Ribbeck, 2015). An ordered biological hydrogel coat is formed by the crosslinking of MUC1, MUC5B, MUC7 and other salivary proteins on oral epithelium as part of the defensive pellicle layer (Hannig, Hannig, Kensche & Carpenter, 2017). The extracellular domain of MUC1 modulates cell to cell and cell to extracellular matrix interactions whereas the cytoplasmic domain is involved in cell signalling (van Putten & Strijbis, 2017). The extracellular, transmembrane and intracellular domain functions of transmembrane mucin 1, MUC1 are outlined in Table 1.. ro of. Mucins contain multiple domains arranged in a specific order to facilitate their functions. The SEA domain is present in membrane bound mucins and has been suggested to be involved in cell–extracellular matrix interactions. There is an epidermal growth factor (EGF) domain. present extracellularly in membrane bound and secreted proteins; its function is to potentiate. -p. proliferative signalling. All membrane anchored mucins have a cytoplasmic tail; this can. undergo posttranslational modifications and interact with proteins to enhance cell signalling. re. (Kumar S et al, 2017).. The fact that MUC1 contain a heterodimeric SEA domain adjacent to the membrane. lP. anchored helical structure suggests that a biological function may be coupled with dissociation (Pichinuk et al, 2012). The encoded dissociation is achieved by mechanical stress rather than chemical or thermal stress. One possible function of the SEA domain at mucus. na. anchoring points is to ensure the dissociation of SEA which allows for mucin shedding and protection of the apical cell membrane of the epithelial cells from rupture. Furthermore, the dissociation of the SEA domain may transmit a signal via some protein ligand that only. ur. associates with an intact SEA and this ligand will be released after SEA has been dissociated from the MUC1 complex (Macao, Johansson, Hansson & Hard, 2006). With respect to the. Jo. two most widely studied isoforms of MUC1 (MUC1/X & MUC1/Y), MUC1/X has been found to mimic the SEA domain in its extracellular domain in that it undergoes autoproteolysis. MUC1/Y is identical to MUC1/X except that in the N-terminal region, there is a SEA domain that does not undergo cleavage. This suggest that an intact SEA module is required for proteolytic cleavage (Kumari & Sudandiradoss, 2013; Sousa et al, 2016). MUC1 is considered to be a highly specific signalling receptor; its function is to monitor the mucosal lumen and initiate epithelial responses through its many domains. The ectodomain.

(9) of MUC1 acts as anti-adhesive since it has a minimum number of tandem repeats to interfere with cell surface receptors. In some instances, MUC1 supports cell attachment by binding selecting ligands (Pamela, Brian, Neeraja & Carson, 2011). The cytoplasmic tail of MUC1 is tyrosine phosphorylated after binding with erythroblastic leukemia viral oncogene homologous (ERBB) ligand and it can modulate a wide array of signal transduction events including modulation of gene transcription, complexing with various cytoplasmic signalling molecules and conferring protection of cells from apoptosis (Carraway, Ramsauer, Haq & Carraway, 2003). Another property of MUC1 is its protective function with regard to the immune cells. It has been demonstrated that MUC1 is induced in T cells by interleukin-7 and. ro of. it becomes polarized at the site of T cell–dendritic cell synapses (Vasir B et al, 2005). Though poorly understood, the effect of transmembrane mucins and their shed fragments on immune system is an interesting area of research. Experiments relating to MUC1 as a therapeutic target in cancer have focused on the extracellular domain but recently the. -p. cytoplasmic domain has been targeted since this part is thought to activate multiple signalling pathways leading to the progression of cancer. Blockade of these pathways could result in. re. inhibition of tumor growth and tumor regression. Therapeutic agents targeting MUC1 are under trial but to date they are not approved for general use (Papadimitriou, Bruchell, Graham & Beatson, 2018). MUC1 expression increases in the tumor tissue and the diverse. lP. nature of tumor cells leads to the exposure of multiple epitopes of membrane bound mucins on the cell surface at the same time representing a potential for effective therapeutic targeting. na. (Pamela et al, 2011). 4. MUC1 & Microplicae. ur. An advanced microscopic study revealed that the superficial cells of the partially keratinized and non–keratinized epithelium are present in the ridge-like folds of plasmalemma, which provide intercellular interdigitation and desmosomal adhesion with adjacent cells. These. Jo. plasmalemma folds were found to be different from the microvillous projections and were termed as microplicae (MPL) (Sperry & Wassersug, 1976). MPL are characteristically present in certain tissues e.g. conjunctiva, cornea, lining oral mucosa, pharynx, esophagus, the intermediate zone of the anal canal and the body of filiform papillae of the tongue (Andrews, 1976; Kullaa, 1986). The structural complexity of microplicae of the superficial cells in the oral mucosa is evident when viewed through a scanning electron microscope..

(10) MPL can be 1) Straight and parallel 2) curved and branched 3) honeycombed or pitted (Asikainen et al, 2015). The MUC1 project from the tips of MPL of oral epithelial cells about 15-30nm above the cell surface. Immunoelectron microscopy has revealed MUC1 binding to MPL of oral epithelial cells and also to keratin inside epithelial cells. The extracellular and intracellular domains of MUC1 provide a tight attachment at the cell surface (Sirviö, Mikkonen, Koistinen, Miinalainen & Kullaa, 2019). The presence of MPL at the apical surface of the oral epithelium provides a surface over which the salivary mucosal pellicle forms and MUC1 has been shown to be prominent in the mucosal pellicle. The oral epithelium produces MUC1. other salivary mucins and proteins (Ukkonen et al, 2017).. ro of. that acts as an anchoring protein for the mucosal pellicle due to its ability to crosslink with. MUC1 is one component of the Oral Mucosal Barrier Complex (OMBC), providing a. signalling pathway between saliva and mucosal cells while stabilizing the mucosal pellicle. -p. (Asikainen et al, 2012). In ocular tissue, a destruction of MPL structures leads to a. dysfunction of the protective layer and results in dry eyes (Gipson, 2007). In analogy to the. re. destruction of MPL in ocular disease, its occurrence in the mouth could lead to a dysfunction in the OMBC; understanding the underlying mechanism might provide insights into the. lP. pathogenesis of several oral mucosal diseases.. 5. MUC1 in normal oral mucosal epithelium. na. The differential staining and intensity pattern of MUC1 in different structures and in different cells suggests that there are differences in the presence, quality and quantity of exposed mucin epitopes on the cell surface. The ducts of the minor salivary glands displayed. ur. immunohistochemical staining of transmembrane mucin MUC1 in cytoplasm and cytoplasmic membranes (Sengupta et al, 2001). The oral stratified squamous epithelium. Jo. showed that MUC1 expression was restricted only to the superficial cell layer of epithelium (Kullaa, Asikainen, Herrala, Ukkonen and Mikkonen, 2014). This restriction of MUC1 to the superficial surface implies that there is a late occurrence of expression of the MUC1 gene product in the differentiation process of oral epithelial cells (Hori, Sugiyama, Soma & Nishida, 2007; Kirkeby, Moe & Bardow, 2010). There is convincing evidence for the production of MUC1 on the superficial surface of oral mucosa (Cleaton, 1975). As is typical of exocrine secretion, MUC1 is produced by rough endoplasmic reticulum, modified in Golgi apparatus and then transported to the apical.

(11) surface. The localization of MUC1 into the small vesicles near the plasma membrane and in the plasma membrane of the cells can be observed in transmission electron microscopy (Ukkonen et al, 2017). MUC1 is produced before the oral cells reaches the apical and/or superficial layer and is expressed on the tips of MPL (Sirviö et al, 2019). In normal oral mucosa, a membranous and cytoplasmic staining of MUC1 can be detected due to its large extracellular and short transmembrane and cytoplasmic domains, which are also the structures mainly accounting for its adherence to oral mucosa (Croce et al, 2007). Both the apical and basolateral localization of MUC1 is required for correct signalling, with basolateral ordered signals being dominant over apical signals. There is some understanding. ro of. of the mechanisms involved in basolateral signalling, but apical signalling has proven difficult to elucidate because it involves coordinating information originating from the. various regions present in cytoplasmic domains of the protein (Folsch, Mattila & Weisz,. 2009). The cytoplasmic domain of MUC1 does not participate in apical localization, instead. -p. two motifs or cysteine residues are involved. One of these residues is located in the extracellular domain and the other is located at the junction of transmembrane and. re. cytoplasmic domains (Pemberton LF et al, 1996). The apical localization of MUC1 in epithelial cells might be involved with sensors on the cell membrane, cell membrane proteins and lipids, glycosylation and the actin cytoskeleton (Weisz & Boulan, 2009). Glycosylation. lP. of specific sites on a protein are often required to mediate apical sorting. The N-terminal region of the extracellular domain of the O-glycosylated part is sufficient to direct apical localization, the intracellular C-terminal region is not involved in this process (Jacob,. ur. na. Alfalah, Grunberg, Odendorf & Naim, 2000).. Jo. 6. Implications of MUC1 for different oral disorders 6.1 Decreased salivary secretion \ Xerostomia The oral mucosal lesions can be attributed to an alteration in the oral defence mechanism mainly in MUC1. Decreased salivation and low levels of MUC1 may lead to oral mucosal diseases (Chang et al, 2011). Furthermore, the transmembrane mucin, MUC1, is considered to be a second line of defence by acting as a sensor towards disturbances and signalling crucial information to the interior of the cell. The reduction in the amounts of MUC1.

(12) subsequently fails to provide a scaffold for the assembly of MUC5B and MUC7 (Tabak, Levine, Mandel & Ellison, 1982; Osailan, Pramanik, Shirodaria, Challacombe & Proctor, 2011). The altered anti-bacterial activity and physical properties of saliva can result from a decrease in shedding of the extracellular domain of MUC1. The extracellular N-terminal alpha subunit and the intracellular C-terminal beta subunit are two subunits of the MUC1 protein. MUC1 is synthesized as a single polypeptide chain which is autoproteolytically cleaved due to the molecular stress into two subunits (α and β) in the endoplasmic reticulum soon after synthesis and is present on the cell surface as a heterodimer. In the cell membrane, the α and β subunits bind together through a strong non-covalent interaction. As the. ro of. extracellular α and β subunits are not covalently bound, they are free to dissociate and rebind for example in a ligand-receptor interaction (Levitin F et al, 2005). Hence, this reduction in. both the first and second lines of oral cavity’s defence system can have serious consequences for the integrity of the oral mucosa.. -p. 6.2 Sjögren syndrome. Sjögren syndrome (SS) is a chronic autoimmune disease, with the loss of epithelial cell. re. homeostatic function in salivary and lacrimal glands causing clinical symptoms such as dry eyes and dry mouth. A histological evaluation can reveal the characteristic sign of peri-. lP. lymphocytic infiltration with germinal center formation and a focal area of lymphomonocytic aggregates. MUC1 has been studied extensively in attempts to clarify its role in the pathophysiology of dry eyes (Caffery et al, 2010). As dry eyes cause changes in the normal. na. ocular environment; these may be attributable to shedding of exposed extracellular portion of MUC1 which in turn signals the ocular epithelial cells to produce more MUC1. On one hand, the increase in expression of MUC1 in dry eyes could be an attempt to protect the healthy. ur. ocular surface on one hand but on the other hand, it may be directly proportional to the severity of the disease. In comparison with the ocular surface, the MUC1 on the oral mucosal. Jo. surface can have a dual role 1) As the disease severity increases, MUC1 expression increases as a compensatory or protective mechanism, 2) the inflammatory signals induced by cytokines upregulate the MUC1 gene expression in either autocrine or paracrine manners, thereby stimulating more than one signal transduction pathway (Li, Wang, Nunes, Troxler & Offner, 2003). There is overexpression and aberrant localization of MUC1 observed in labial salivary gland of SS patients (Barrera et al, 2012). The pro-inflammatory cytokines and epigenetic factors.

(13) are responsible for the MUC1 gene overexpression. In SS patients, there is marked apical – basolateral re-localization of proteins with a marked aberrant accumulation of secretory granules throughout the cytoplasm (Prez et al 2010). The presence of secretory granules and extracellular matrix for mucins indicates that ectopic exocytosis may be due to 1) alterations in the cytoskeleton (actin microfilament), 2) changes in the secretory pathway (Rab GTPase – Ras superfamily of small G protein; guanosine triphosphate hydrolase enzyme) involved in membrane recognition and fusion of vesicles, 3) alterations in membrane fusion receptors (SNARE proteins – soluble N-ethylmaleimide – sensitive factor attachment protein receptor) (Wang et al, 2008). These changes result in a disturbed mucin distribution and its secretion. ro of. by ductal and acinar cells of the minor salivary glands explains the cytoplasmic accumulation of MUC1 in SS patients (Prez-Vilar, 2007; Culp, Stewart & Wallet, 2019). 6.3 Irradiated tissue / Irradiation effect on oral mucosa. Oral cancer treatment involves various types of adjuvant and neoadjuvant therapies to combat. -p. the disease as well as avoiding dose-limiting, toxic side effects. Radiotherapy is associated with a significant increase in cure rates for many oral malignancies but inevitably involves. re. exposure and damage to healthy surrounding tissues (Pandya et al, 2014). As a consequence, the oral mucosa, skin, maxilla, mandible and salivary glands undergo several undesirable. lP. reactions. Mucositis, candidiasis, dysgeusia, fibrosis, radiation caries, periodontal destruction, soft tissue necrosis, osteoradionecrosis, trismus, and xerostomia-like complications are often observed in irradiated patients (Stone, Coleman, Anscher & McBride, 2003; Tolentino et al,. na. 2011).. How does radiation alter the physiology of MUC1 and microplicae? Radiation causes damage. ur. in different layers of epithelium, cell junctions, alterations in cell-extracellular matrix interactions and also penetrates into the underlying deeper connective tissue. Irradiation therapy induces structural abnormalities in the MPL, disrupting the epithelial homeostasis in. Jo. superficial and deeper layers (Asikainen et al, 2017). MUC1 is expressed on the tips of MPL of superficial cells, any alteration in the MPL structure may affect the expression of MUC1, resulting in a loss of its anti-adhesive and anti-bacterial properties. This can result in oral mucositis and ulceration of oral epithelium (Mukherjee, Philip, Coates, Lorimore & Wright, 2014). The increase in the variety of cell surface patterns encountered after radiation therapy, implies that irradiation affects the internal structure of the cell and only later are there.

(14) changes in the surface morphology on the superficial surface. Prior to the radiation, the MPL appeared to be straight and parallel, during a course of low dose radiation it was found to be straight, parallel, branched or unbranched. With increasing doses, the MPL showed a loss of the straight parallel form in some areas to become short parallel ridges with a, honeycombed appearance; in few areas, the MPL was non-parallel, with a circular pattern and a thick short discontinuous shape (Robertson, Wilson, Wilson, Carr & Hunter, 1987). The difference in the pattern of microplication after various doses might alter the adherence of MUC1 at the cell surface. Hence, exposure to radiation increases the exfoliation of superficial cells due to their poor adhesion, disruption in junctional complexes, thereby exposing the underlying deeper. ro of. epithelial cells with their different MPL patterns (Southgate, Williams, Hodges & Trejdosiewicz, 1987).. The constant exposure of the mucous layer to varying doses of radiation was reported to provide a base to allow bacteria to adhere onto cellular surfaces and this could clinically. -p. correlate with the pain and discomfort associated with irradiated mucositis. An Fourier Transformed Infrared Spectroscopy (FTIR) study revealed structural and biochemical. re. changes in the superficial layer of the oral mucosa (Ukkonen et al, 2019). Although we now have some knowledge about the relationship between MPL and MUC1, there are a few areas which have remained elusive and needs exploration – Does the varying pattern of the MPL. lP. affect the stability of MUC1 at the cell surface? Does the pattern of the MPL indicate the degree of differentiation of oral epithelial cells? Whether morphometric analysis of MPL. na. provide a basis for understanding the progression of disease? 6.4 Oral inflammation. ur. There is convincing evidence supporting the multifunctional involvement of mucins in complex biological processes like epithelial cell renewal, differentiation, cell adhesion and cell signalling (Carraway, Ramsauer, Haq & Carraway, 2003). The constant exposure of oral. Jo. epithelial cells to virulent and commensal microbes or their secreted products leads to increased transcription of MUC1 by pro-inflammatory mediators (Li et al, 2003). These proinflammatory cytokines are known to upregulate MUC1 expression in several epithelial and hematopoietic cell lines (Reddy et al, 2003). There are two primary types of cytokines involved in the regulation of the MUC1 gene: 1) Type I cytokines such as interleukin (IL) 2 & 12, interferon gamma (IFN-γ) and tumor necrosis factor (TNF), which elicit a cellular immune response, 2) Type II cytokines.

(15) including IL – 4, 5, 6, 9, 10 & 13, evoke a humoral immune response. These cytokines interact either between themselves or with other biologically active compounds to develop a synergistic mucin induction. It is known that there is a tightly regulated cross-talk between various signalling molecules which either directly or indirectly regulates the expression of MUC1 (Andrianifahanana, Moniaux & Batra, 2006). The anti–inflammatory activity of MUC1 can be evoked by suppressing the activation of Toll–like receptors (TLR) (Ueno et al, 2007). There are several TLRs both at the cell surface and in the cytoplasm involved in the process of endocytosis. Once activated, either by bacterial, viral, fungal or other by-products, they eventually activate the Nuclear factor kappa. ro of. B ligand (NF-κB). MUC1 suppresses the activation of the NF-κB ligand induced by the TLRs, thereby preventing the release of pro-inflammatory products and cytokines (Akira &. Takeda, 2004). The cytoplasmic domain of MUC1 is involved in cell signalling as it contains multiple motifs for crucial proteins including Phosphoinosotide 3-kinase (PI3K), Shc adaptor. -p. protein (Shc), Proto-oncogene tyrosine protein kinase (Src), β-catenin, Glycogen Synthase Kinase 3 beta (GSK - 3β), Erythroblastic leukemia viral oncogene homologue (ErbB). re. receptor family and Growth factor receptor bound protein 2 (Grb-2). Hence, it is possible that the anti-inflammatory action of MUC1 is mediated through some or all of these pathways that interact with the common TLR signalling pathway (Singh & Hollingsworth, 2006).. lP. (Figure 1). An in vitro study on human ocular surface epithelial cells suggested that the inflammatory. na. cytokines influence mucin expression and ectodomain release for cell signalling. The data revealed various patterns through which the regulation of mucin production can be changed; 1) the pattern of expression, protein biosynthesis and ectodomain release are upregulated in a. ur. sequential way mainly by the effect of TNF-α and INF-γ cytokine. This affects the production and turnover of MUC1 at the transcriptional level. 2) Another pattern reflected the. Jo. independent regulation of the activity of the release of the MUC1 ectodomain, leading to a change in total cellular protein (Albertsmeyer, Kakkassery, Spurr-Michaud, Beeks & Gipson, 2010; Hori et al, 2007). Considering the crucial role of MUC1 in inflammation, we postulate that a) measurement of cytokine levels and ectodomain release levels might provide an index for assessing the extent of inflammation on the surface of epithelial cells, and b) epithelium grown in vitro without the possibility of undergoing stromal interactions might respond differently from healthy oral epithelial tissue i.e. the later should be the preferred study cell system..

(16) 6.5 Oral precancer and cancer Oral precancer and oral cancer involve approximately two thirds of the population living in the developing countries. The diverse nature of the disease is reflected in the dysregulation of multiple pathways linked to cellular differentiation, cell cycle control, apoptosis, angiogenesis, and metastasis. This could result from the accumulation of multiple genetic alterations e.g. either by some genetic predisposition or by environmental exposures, including tobacco, alcohol, chronic inflammation and viral infections. MUC1 has been considered to be a useful indicator due to its increased rate of expression as the malignancy changes from oral precancer to cancer (Kumar et al, 2016). Up-regulation of MUC1 is. ro of. reflected in early cellular changes such as altered cell-to-cell or cell-to-matrix interactions causing reduced adhesion, change in invasive capacity and an increased metastatic potential of tumor cells (Yamada et al, 2011; Hayry et al, 2018).. How does MUC1 influence tumor growth and metastasis? Various mechanisms have been. -p. proposed to explain how MUC1 promotes tumor growth. The structure and distribution of. cell surface glycoproteins are thought to influence the biologic behavior of tumor cells during. re. malignant transformation and tumor progression. For example, MUC1 increases the expression of Vascular Endothelial Growth Factor (VEGF), tumor angiogenesis and tumor. lP. growth via activating Insulin like growth factor 1 receptor\ protein kinase B (IGF – 1R/ AKT) pathway in vivo (Woo et al, 2012). MUC1, usually does not promote any transcriptional activity, but does promote endothelial cell migration and proliferation by activating AKT and. na. Extracellular signal regulated kinases (AKT and ERK1 /2), which are known to be proangiogenic target gene promoters. IGF-1R is an important energy metabolism and growth regulator and also a potent and growth factor; its functional effects are mediated through. ur. PI3K, AKT and ERK1 /2. MUC1 regulates IGF-1R during tumorigenesis either directly or indirectly by ultimately increasing the expression of VEGF and thus it can contribute to. Jo. angiogenesis (Ping, 2015; Isayeva et al, 2015). MUC1 acts as an antiadhesion molecule, and its expression increases during the progression from oral premalignancy to malignancy (Croce, Rabassa, Pereyra & Segal-Eiras, 2008). The invasion or metastasis of cancer cells is due to disturbed glycoconjugation and this process can be linked to transformed cellular adhesion. The cell-to-cell and cell-to-matrix adhesion becomes reduced due to overexpression of MUC1 because either the large, elongated or rigid structure of MUC1 interferes with its interaction with adhesion molecules and its ligands or.

(17) there are reduced levels of interaction between integrins and the extracellular matrix (Nitta, Sugihara, Tsuyama & Murata, 2000). It seems that the degree of cellular motility correlates with the increased expression of MUC1 and the ability of these malignant cells to escape immunosurveillance. The exposure of the MUC1 epitope on the cell membrane may prevent an interaction between immune cells and tumor cells, resulting in decreased activity of other immune cells i.e. natural killer cells and tumor cells, thereby allowing the malignant cells to successfully manage to escape the immune surveillance (Boldrup et al; 2017; Kamikawa et al, 2015). The features of survival, escape and invasion are unique characteristics of malignant cells and. ro of. this includes the involvement of MUC1. Its cytoplasmic tail is responsible for various forms of cell signalling, e.g. through binding to beta-catenin and mitogen activated protein kinase (MAPK), resulting in the initiation of the epithelial-mesenchymal transition, a condition. known to promote metastasis (Nitta et al, 2000). One physical property of MUC1, i.e. the. -p. presence of sialic acid on the terminal carbohydrate structure, might be more important in. determining the extent of metastasis, as sialylation of this part of the cell surface imparts a. re. strong negative charge that affects invasion and metastasis (Shobhita et al, 2018). MUC1 also performs novel regulatory functions in subcellular protein trafficking e.g.. lP. regulating the nuclear activity of Epithelial growth factor receptor (EGFR), a process that changes in cancer cells. In the presence of MUC1, EGFR stimulation takes place at the cell surface and the complex is rapidly endocytosed by caveolae/caveolin 1. Retrograde. na. trafficking of MUC1 and EGFR takes place with the complex being imported to the nucleus. EGFR is associated with cyclin D1 to promote active transcription whereas MUC1 helps in recycling EGFR back to the cell membrane (Bitler, Goverdhan & Schroeder, 2010). The. ur. expression of EGFR and cyclin D1 are increased in oral cancer, triggering a loss of cell adhesion and the initiation of cell proliferation thereby resulting in tumor growth, invasion. Jo. and metastasis. The signal transduction cascade begins at the cell surface with intracellular signalling proteins being transferred first to cytosol and then to the nucleus (Figure 2). MUC1 expression in cancers allows EGFR to bind to the Gene encoding cyclin D1 protein (CCND1) promoter, to upregulated cyclin D1 which increases proliferation and genetic instability within the cell, thereby initiating tumorigenesis (Kalish et al, 2004). 6.6 Salivary gland tumors.

(18) Salivary glands tumors account for approximately 3% of all head and neck malignancies (Handra-Luca, Lamas, Bertrand & Fouret, 2005; Sessions, Harrisson & Forastiere, 2000). The prognostic implications of the major salivary gland tumors have been extensively studied; instead there are very few investigations of the minor salivary gland tumors. MUC1 has been evaluated cytologically, histologically and immunohistochemically in some of the benign (e.g. pleomorphic adenoma, Warthin’s tumor, cystadenoma) and malignant (e.g. Mucoepidermoid carcinoma, adenoid cystic carcinoma, acinic cell carcinoma, pleomorphic ex carcinoma) tumors in attempts to assess the biological and prognostic behavior of the tumors (Karbnova et al, 2014; Kasafuka et al, 2008; Mannweiler, Beham & Langner, 2003,. ro of. Matse, Bharos, Veerman, Bloemena & Bolscher, 2017; Lanzel, Pourian, Sousa Melo, Brogden & Hellstein, 2016; Terada, 2013). The apparent heterogeneity of binding of. monoclonal antibodies is due to epitope masking by glycosylation of MUC1; this thought to be due to the O-glycans which are attached to the threonine of the mucin derived peptide. antigen (PDTRP) epitope itself or to serine or threonine residues in the vicinity of the mucin. -p. derived epitope (Cao & Karsten, 2001).. re. In malignancies, MUC1 is redistributed from luminal membrane of normal polarized duct cells to the entire surface of non-polarized tumor cells. In tumor cells, MUC1 has fewer, shorter and less branched glycans in contrast to its structure in normal cells i.e. elongated and. lP. highly branched glycans. The hypoglycosylation of MUC1 in tumor cells impacts on its stability at the cell surface and the subcellular localization of the protein increases intracellularly via endocytosis (Lloyd, Burchell, Kudryashov, Yin, Taylor-Papadimitriou,. na. 1996). Hence, the cell phenotype can be determined immunohistochemically by determining MUC1, its complete absence can help in identifying indolent tumors (Handra-Luca et al,. ur. 2005). Salivary gland tumors show a greater tendency towards proliferative and mucosecretory activity. In mucoepidermoid carcinoma, the most common malignant tumor,. Jo. MUC1 expression was found to be independent of Ki-67 (proliferative marker) expression (Goncalves et al, 2011). MUC1 expression was shown to increase with the increasing histological grades of the tumor indicating a worse prognosis, a higher rate of recurrences, metastasis and a decrease in disease-free intervals. MUC1 plays a coordinated role with p120 catenin (a protein which mediates cadherinmediated cell adhesion and actin cytoskeletal organization) in regulating cell adhesion, motility and metastasis (Liu et al, 2014). The binding of MUC1 with p120 catenin facilitates E cadherin trafficking to the cell surface as described by several authors while studying other.

(19) cancerous cells (Bitler et al, 2010; Singh & Hollingsworth, 2006). MUC1 overexpression occurs due to several mechanisms including increased transcription, amplification of MUC1 genomic locus or loss of post-transcriptional regulation. 7. Conclusion MUC1 has been the most widely studied mucin due to its large glycosylated structure and varied functions performed at the cell surface. Aberrations in MUC1 expression are often observed in human oral cancers, the extent of expression increases from inflammatory to noninvasive to invasive lesions. The changing views of their function in oral mucosal physiology and pathology can be traced to our improved understanding of the complexity of the cell. ro of. membrane and its associated surface structures. Hence, novel strategies have to be designed to clarify the mechanistic aspects of MUC1 expression as well as finding novel ways to. rectify the abnormal MUC1 expression often observed in oral cancers. Still today, there. remains an unanswered question of whether the deregulation of MUC1 expression is a cause. -p. or a consequence of the transforming phenotype. It is now essential to appreciate that MUC1 is a major part of a multimolecular signalling platform, and that it may be a potential target. lP. re. for novel therapeutics in the future.. Jo. ur. na. Conflict of Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper..

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(29) Jo. ur. na. lP. re. -p. ro of. Figure Captions:. Figure 1: MUC1 mediated anti-inflammatory signalling pathways..

(30) ro of -p re lP. na. Figure 2: Operational role of Epithelial growth factor receptor (EGFR) in the. Jo. ur. presence and absence of MUC1..

(31) Table Legend: Table 1 – Functions of the domains of the transmembrane mucin MUC1.. MUC 1 domains. Physiologic Protective function. Loss of resistance and metastasis. Provide resistance to external stimuli. Hindrance in cell to cell contact. Dampening of immune responses. Activation of epithelial growth. by receptor shielding. factor receptor. Decoy receptor for invasive pathogens. Cancer progression. ro of. Extracellular domain. Pathologic. Resistance to drugs. Transmembrane domain. Phosphorylation of attached intra cellular domain. and cytokine secretion. EGF receptor interaction Cell proliferation and hampering. Regulation of cell proliferation and. apoptosis. lP. na. Signal transduction. ur Jo. cadherin/β-catenin. Inflammasome regulation. apoptosis. Cytoplasmic domain. re. Variation in NF-κB pathway. Cell-cell contact through E-. -p. Activation of inflammatory pathways. Phosphorylation of cytoplasmic tail. Regulation and interaction of various transcriptional factors..

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