MUCINS AND TOLL-LIKE RECEPTORS: KITH AND KIN IN INFECTION AND CANCER

Abstract

Inflammation is underlying biological phenomenon common in infection and cancer. Mucins are glycoproteins which establish a physical barrier for undesirable entry of foreign materials through epithelial surfaces. A deregulated expression and an anomalous glycosylation pattern of mucins are known in large number of cancers. TLRs are class of receptors which recognize the molecular patterns of invading pathogens and activate complex inflammatory pathways to clear them. Aberrant expression of TLRs is observed in many cancers. A highly orchestrated action of mucins and TLRs is well evolved host defence mechanism; however, a link between the two in other non-infectious conditions has received less attention. Here we present an overview as to how mucins and TLRs give protection to the host and are deregulated during carcinogenesis. Further, we propose the possible mechanisms of cross-regulation between them in pathogenesis of cancer. As both mucins and TLRs are therapeutically important class of molecules, an understanding of the underlying molecular mechanisms connecting the two will open new avenues for the therapeutic targeting of cancer.

1.0 Introduction

Mucins (MUCs) and Toll-like receptors (TLRs) are essential in giving protection to the host and maintaining cellular homeostasis [1]. MUCs are high molecular weight glycoproteins produced and mostly distributed between epithelial tissues lining ducts and lumens where they provide protection and lubrication [2]. Akin to their role under physiological conditions, cancer cells exploit MUCs in protecting themselves under adverse conditions [3]. An aberrant expression and glycosylation pattern of MUCs are thus observed in many carcinomas [3]. Though the precise molecular mechanisms regulating the expression of MUCs are areas of investigation nonetheless many inflammatory cytokines and transcription factors are known to regulate their expression [4–7]. In the present article we focus on the interrelationship between MUCs and TLRs, as TLRs initiate inflammatory cascade on recognition of specific pathogen-associated molecular patterns (PAMPs) and consequently they may be important regulators of MUC expression.

TLRs are type 1 membrane anchored receptors, which recognize and regulate immune responsive genes [8, 9]. Deregulated TLR activation is observed in a number of pathological processes [10]. TLRs also recognize a large number of ‘danger signals’ which are often host-derived molecules [11] and heterogeneous tumour microenvironment is a rich source of such endogenous molecules which are recognized by TLRs. Hence there is consistently higher TLR expression in several cancers [12, 13].

The present review article provides an overview to the biology of MUCs and TLRs and discusses how they are regulated during microbial infections. We propose the possible molecular mechanisms of cross-regulation between MUCs and TLRs in cancer. Several members of MUC family are in clinics as important diagnostic and prognostic markers [3]. Identification of novel approaches to modulate the expression of MUCs has promising therapeutic implications. As TLRs have gained remarkable clinical significance [14, 15] in only two decades of their discovery, an understanding of the molecular mechanisms of cross-regulation of MUCs and TLRs will help us with new opportunities to fight against cancer.

2.0 Biology of MUCs

MUCs are a family of large glycoproteins which represent the major structural components of the mucus [2, 16]. The basic structure of a MUC molecule consists of a protein backbone, known as ‘apomucin,’ which is attached with a large number of O-linked and a few N-linked oligosaccharides. A striking feature of MUCs is the presence of VNTR (variable number tandem repeats) [2, 17, 18]. There are 20 mucin genes identified which have wide tissue distribution and specificity [19, 20]. MUCs can be grouped into two different categories based on their sub-cellular localization - secreted and membrane-bound. The distinctive feature of membrane-bound MUCs is the presence of a transmembrane domain which attaches them to the plasma membrane [2, 17]. Secreted MUCs are produced from the surface epithelium by specialized ‘goblet cells’ and ‘mucous cells’ from sub-mucous glands [2] and include MUC2, MUC5AC, MUC5B, MUC6–8 and MUC19. They have characteristic cysteine-rich regions termed as ‘CK’ (Cysteine Knot; site for mucin dimerization) and ‘D’ (site for oligomerization) domains [21, 22]. The membrane-bound MUCs include MUC1, MUC3, MUC4, MUC12–17 and MUC20. They are type I membrane-anchored proteins with trans-membrane domain an NH₂-terminal extra-cellular region and a COOH-terminal intracellular cytoplasmic tail [2, 23]. Membrane-bound MUCs can also be released from the plasma membrane by enzymatic cleavage and alternative splicing [2]. Some MUCs such as MUC1 and MUC4 have more wide tissue distribution as compared to MUC2 which is primarily localized in the intestines [23]. The characteristic extensive post-translational modifications in MUCs such as glycosylation, sialylation and sulfation in a cell-type specific manner gives them specialized structure and facilitates their interaction with the extracellular and membrane bound proteins [24].

2.1 Physiological functions of MUCs

MUCs form a gelatinous layer on the apical surface of the epithelial cells by serving as a physical barrier between the lumen and epithelium which is continually exposed to microbial pathogens [2, 25]. Further any pathogenic bacteria which might have escaped is entrapped and eliminated by the dense mucin matrix [25]. The extensive glycosylation of MUCs confers them the property of hydration and lubrication by providing a hydrophilic environment [3]. The TR region of MUCs is particularly rich in neutral and charged oligosaccharide structures which regulate the movement of molecules. MUCs gels hold biologically active molecules such as trefoil factors (TFFs) [26] which aid them in providing protection to the epithelial surfaces and promote wound healing [3, 26, 27]. Further MUCs play critical roles in cellular events influencing key functions of proliferation, differentiation, transformation and adhesion [2].

3.0 Biology of TLRs

TLRs are membrane receptors which upon recognition of pathogen-associated molecular patterns (PAMPs) initiate and activate the immune response against invading pathogens [9, 28] and till date 10 TLRs have been identified in humans. TLRs1–9 are well characterized and conserved between mice and humans. TLR10 is present in humans but not in mice whereas murine TLRs11–13 remains ambiguous. [29]

TLRs are homologous to Drosophila Toll protein (and thus named ‘Toll-like receptors’) and human interleukin-1 receptor (IL-1R) family [30]. In Drosophila, TLRs are involved in the embryonic development and defence against microbial infections [31–33] whereas in mice the only defects of TLR knock out are related to immune dysfunction [34]. Upon infection Drosophila Toll protein recognizes the endogenous protein Spatzle which is produced as a result of self-proteolytic cleavage of pro-Spatzle [35] while mammalian TLRs directly bind to PAMPs. The evolution of mammalian TLRs to recognize the molecular patterns gives multiple advantages to the host as PAMPs are differentially expressed molecules generally essential for the survival of the microbe and also enable the host to recognize and mount an immune response tailored against a specific class of microbe [36–38]. As TLRs recognize unique patterns in microbes they are known as pattern recognition receptors (PRRs). Other classes of PRRs are receptor kinases, mannose receptors, NOD-like receptors and RNA helicases, C-type lectin receptors [29, 39, 40].

Structurally TLRs may be divided into extracellular and cytoplasmic domains. The extracellular domain consists of 19–25 tandem leucine rich repeat (LRR) which is 24–29 amino acids in length and is believed to interact directly with the ligands [41]. The cytoplasmic domain of TLRs is similar to IL-1R and is known as the Toll/IL-1 receptor (TIR) domain [42]. TLRs are broadly expressed on immune cells and epithelial lining of cells at sites which are continually exposed to invading pathogens [43]. As the primary function of TLRs in mammals is defence, TLRs are also expressed in several types of cells which form the first protective barrier to invading pathogens such as cells of the skin, respiratory, intestinal and genitourinary tracts that form the first protective barrier to invading pathogens [44].

Quite remarkably, the sub-cellular localization pattern of TLRs in the cells has also evolved to facilitate the microbial ligand recognized by them. TLRs which recognize outer cell wall components of bacteria and fungi are expressed on the cell surface (TLR1, -2, -4, -5, -6, -10) whereas TLRs involved in the recognition of nucleic acid components are distributed in the intracellular compartments (TLR3, -7, -8, -9) [37, 45]. Recently a number of chaperones such as gp96, PRAT4A and Unc93B1 have been identified to play critical role in TLR localisation [46]. The salient feature of TLR ligands are their enormous molecular diversity which continues to grow but the most studied ligands for TLRs remain those from the microbial origin. The principle ligands for TLR2 in combination with TLR1 or TLR6 are bacterial cell wall components such as lipoproteins, lipotechoic acid and fungal zymosan. TLR4 is activated by lipopolysaccharide from gram-negative bacteria and TLR5 by bacterial flagellin. The intracellular TLRs such as TLR3 is activated by the double-stranded RNA, TLR7 and TLR8 by single-stranded RNA and TLR9 by unmethylated CpG DNA motifs [9, 28]. In addition to the microbial ligands, there are host-derived endogenous molecules which signal the presence of ‘danger’, aptly called as danger-associated molecular patterns or DAMPs [11]. A large number of endogenous ligands have been identified as shown in the table 1. Very recently, the mitochondrial DNA which shows resembles to bacterial DNA due to the presence of non-methylated CpG motifs has been shown as endogenous TLR9 ligand [47]. However the theory of endogenous ligand recognition is challenged suggesting that endogenous ligands only facilitate the presentation of microbe-derived products to the TLRs [48]. The endogenous ligands act as PAMP sensitizing molecules by binding to the PAMPs released from commensal intestinal microbes and lower the cellular threshold for responsiveness to PAMPs [48–50]. Further studies are warranted to dissect the ability of putative TLR ligands in mediating the biological response. In table 1 we summarize the exogenous microbial ligands, host-derived endogenous ligands, sub-cellular localization and cell type expression pattern for TLRs1–11.

Table 1.

TLR ligands, sub-cellular localization and cell type distribution

TLR	Exogenous ligands	Endogenous ligands	Cell types [51]
TLR1	Tri-acylated lipopeptide [52]	Not identified [51]	Basophils, B-lymphocytes, dendritic cells, fibroblasts, keratinocytes, macrophages
TLR2	Peptidoglycan [53] Pam3CSK4 [54], [55] lipopeptide [56] Lipoteichoic Acid [57]	HSP60, HSP70, HSP90 [51, 58] RAGE [59], PAUF [60]	Basophils, B-lymphocytes, dendritic cells, endothelial cells, fibroblasts, keratinocytes, Langerhans’ cells, macrophages, mast cells, NK cells, neutrophils
TLR3	Viral Double stranded RNA, Poly I:C [9, 51]	Endogenous mRNA [61]	B-lymphocytes, dendritic cells, endothelial cells, fibroblasts, keratinocytes, macrophages, NK cells, neutrophils
TLR4	Lipopolysaccride from gram negative bacteria, taxol [9]	HSP60, HSP70, HSP90 [58], extra domain A of fibronectin, β-defensin 2, Fibrinogen [51], heparan sulphate [9] RAGE [59] PAUF [60]	Basophils, B-lymphocytes, dendritic cells, endothelial cells, fibroblasts, keratinocytes, Langerhans’ cells, macrophages, mast cells, NK cells, neutrophils, regulatory T cells
TLR5	Bacterial Flagellin [51]	Not identified [51]	B-lymphocytes, dendritic cells, endothelial cells, fibroblasts, keratinocytes, macrophages, NK cells, regulatory T cells
TLR6	Di-acylated lipopeptide [9]	Not identified [51]	Basophils, B-lymphocytes, dendritic cells, fibroblasts, keratinocytes, macrophages, NK cells, neutrophils
TLR7	Single-stranded RNA (HIV-1) [51]	Imidazoquinolines (Imiquimod, Resiquimod Loxoribine Bropirimine [51]	B-lymphocytes, dendritic cells, fibroblasts, macrophages, neutrophils,
TLR8	Single-stranded RNA (HIV-1) [51]	Imiquimod, Resiquimod[51]	dendritic cells, fibroblasts, macrophages, NK cells, regulatory T cells
TLR9	Unmethylated CpG DNA [9]	Herpes simplex virus-2 [51] Mitochondrial DNA [47]	Basophils, B-lymphocytes, dendritic cells, endothelial cells, fibroblasts, keratinocytes, Langerhans’ cells, macrophages, NK cells, neutrophils
TLR10	Not identified [51]	Not identified [51]	dendritic cells, keratinocytes, B-lymphocytes
TLR11	Uropathogenic bacteria [9]	Not identified [51]	Endothelial cells

3.1 Physiological functions of TLRs

In mammals TLRs are the integral to the functioning of innate immune system, their primary role being to encounter and eliminate invading pathogens [36–38]. Upon binding with the respective ligands, the cytoplasmic TIR domain of TLRs associates with the TIR domain of the adaptor proteins MyD88, TIRAP, TRIF and TRAM. All TLRs except TLR3 signal through MyD88-dependent pathway. TLR3 signals only through MyD88-independent TRIF-dependent pathway while TLR4 is unique as it can signal through both MyD88-dependent as well as MyD88-independent pathways. Upon binding with the TIR domains of the TLRs MyD88 undergoes dimerization which allows for the recruitment of downstream kinases IRAK-1 (IL-1R-associated kinase-1) and IRAK-4 through death domain (DD) homophilic interactions. MyD88 binds to IRAK-4 thereby promotes phosphorylation of critical residues in IRAK-1 by IRAK-4. The downstream events include IRAK-1 phosphorylation and its interaction with TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6), leading to the activation of NF-κB (nuclear factor κB), JUN N-terminal kinases (JNKs), p38 and ERKs (Extracellular signal-regulated kinases) and interferon regulatory factor (IRF3, IRF5 and IRF7) signaling pathways. The activation of TRIF-dependent pathway by TLR3 and TLR4 leads to the activation of IKK complex consisting of TBK1, IKKε/IKKi further leading to the activation of NF-κB and IRF3 [62]. These molecules further lead to the production of pro-inflammatory cytokines and chemokines which activate adaptive immunity and together mount an effective immune response against invading pathogens [63]. Even though the signaling by TLRs operate through a common pathway, the various TLR agonists induce a unique cytokine/chemokine profiles that is best tailored against a particular microbial class [64]. The detailed signaling pathways are indicated in the Figure 1.

Fig. 1.

Toll-like Receptors (TLRs) recognize specialized structural motifs on the microbes known as pathogen-associated molecular patterns (PAMPs). The cellular distribution of TLRs represents the site of their encounter with the pathogens – TLRs which recognize cell wall moieties (TLR-2,-4, -5) are present on the cell-membrane whereas TLRs which recognize nucleic-acids (TLR3, -7, -8, -9) are localized in the endosomal compartment. TLR1, -6 signal through heterodimerization with TLR2 and thereby localized on the cell membrane. Upon ligand binding, the cytoplasmic Toll/IL-1 receptor (TIR) domain of the TLRs associate with TIR domain of the adaptor proteins MyD88, TIRAP, TRIF and TRAM. MyD88 is required for signaling by all TLRs except TLR3 which together with TLR4 signal through TRIF dependent-MyD88 independent pathway. TLR4 is unique in that it utilizes all the four adaptor proteins for signaling. The use of alternative adaptors provides specificity to the TLR signaling pathway. Further MyD88 recruits IRAK4 and IRAK1 molecules to the TLR signaling complex through interaction with their death domains (DD). This leads to the activation of TRAF6 which further activates several effector molecules such as transcription factors NF-κB, IRF3 and MAP kinases like p38, ERK1/2 and JNK. Downstream signaling events by these molecules regulate the expression of pro-inflammatory cytokines and chemokines.

4.0 MUCs and TLRs in infection

Several microbial products up regulate the expression of MUCs and TLRs [65]. Upon recognition of PAMPs the principle receptors of innate immune system TLRs direct the immune response against pathogens [9, 28]. In fact, it has been suggested that TLRs serve as a bridge between innate and adaptive forms of the immunity [38, 66]. A basal low-level expression of MUCs is up regulated on encounter with microbial pathogens. This is particularly evident in the case of intestinal MUCs, which provide several advantages to the commensal bacteria in terms of nutrition and protection [1].

It was observed that Gymnophalloides seoi adult antigen enhances the expression of TLR2 and MUC2 in HT29 cells [65] and treatment with monoclonal antibody to TLR2 abrogates the expression of MUC2 [65]. Mycoplasma pneumoniae has also been shown to induce MUC5AC expression via TLR2 and inhibition of TLR2 and NF-κB reduced the expression of MUC5AC, indicating the role of TLR2 in MUC5AC expression [67]. The outer cell wall component lipoprotein p6 from Haemophilus influenzae (NTHi) induces the expression of MUC5AC via TLR2 signaling pathway [68]. Another study by Kyoung et al. shows LPS induced up regulation of MUC5AC with concurrent increase in TLR4 expression and the knock down of TLR4 with siRNA leads to the decrease in MUC5AC suggests the involvement of TLR4 in LPS-induced MUC5AC gene expression [69, 70]. Streptococcus pneumoniae pneumolysin induce TLR4 dependent MUC5AC mucin transcription in airway epithelial cells which is important to maintain effective mucosal protection against S. pneumoniae infection [71]. Thus it is evident that MUCs and TLRs co-operate to strengthen the mucosal immune response. [72]. Several signaling molecules such as MAP kinases and NF-κB directly regulate MUC expression. Also various cytokines and chemokines via activation of EGF-tyrosine kinase and MAP kinase pathways regulate MUC expression [73]. MUCs also regulate TLR signaling as MUC1 has been shown to negatively regulate TLR5 signaling [74]. The cytoplasmic tail of mucin MUC1 serves as a receptor for P. aeruginosa flagellin which competes with TLR5 binding to its ligand thereby negatively regulating its signaling in airway epithelial cells [74, 75]. Although, there are few studies on the regulation of TLRs by MUCs, in table 2 we have summarized the known interactions between MUCs and TLRs in infection. Given the cross-regulation between MUCs and TLRs in infection we speculate that cancer cells might use the TLR signaling pathways much in the same way to up regulate the expression of MUCs which in turn may also regulate TLR signaling. In the preceding sections we discuss the significance of MUCs and TLRs in cancer and propose the molecular mechanisms of their interaction.

Table 2.

MUC and TLR in infection

Microbe/microbe derived product	TLR	Mucin	Cell Type	Reference
Gymnophalloides seoi	TLR2	MUC2	HT29	[65]
Mycoplasma pneumoniae	TLR2	MUC5AC	A549	[67]
Haemophilus influenza (lipoprotein p6)	TLR2	MUC5AC	HMEEC-1	[68]
LPS	TLR4	MUC5AC	Airway epithelium	[69, 70]
Streptococcus pneumoniae	TLR4	MUC5AC	Airway epithelium	[71]

5.0 MUCs and TLRs in cancer

MUCs are a class of major differentially expressed proteins between normal and cancer cells which makes them a potential target for anti-cancer therapies. As reviewed by Hollingsworth and Swanson [3] the up regulation of MUC expression is advantageous to cancer cells in several ways. In addition to giving physical protection MUCs provide additional advantages by sequestering the growth factors and cytokines. MUCs also promote the invasive and metastatic potential by influencing the adhesive and anti-adhesive cell-surface properties of tumour cells. Thus an altered MUC expression and/or glycosylation help in cancer progression by imparting specific growth advantage to the cancer cells [3]. In several carcinoma and haematological malignancies the abnormal expression of several MUCs are observed. As a class of glycoproteins MUCs are increasingly recognized as potential markers of disease progression and metastasis and are currently investigated as therapeutic targets for cancer [76]. MUC1 is over expressed in many leukaemia and solid cancers. CA15-3 assay, which measures the circulating MUC1-N subunit, is approved by the FDA as a prognostic marker in breast cancer [77, 78]. MUC4 is a prognostic marker for ovarian and pancreatic cancer. CA125 which is derived from a small subunit of MUC16 is in clinical use as diagnostic marker for ovarian cancer [3, 79]. The association between normal physiological expression and aberrant MUC expression in various cancers is summarized in table 3.

Table 3.

MUCs in physiological conditions and aberrant expression in cancers

Mucin	Distribution in normal physiological conditions	References	Aberrant expression/Glycosylation in Cancer	References
MUC1	Salivary glands, airways, oesophagus, stomach, pancreas, duodenum, male and female reproductive tract	[16, 80–86]	Breast, colon, gastric, lung, ovarian, pancreatic, prostate cancer and haematological malignancies, renal	[77, 87–93]
MUC2	Airways, duodenum, colorectum	[80, 84, 94]	Breast, colon, gastric, pancreatic cancer	[91, 93, 95, 96]
MUC3	Salivary, airways, hepatobiliary, duodenum, colorectum	[80, 83, 84, 94, 97]	Breast, colon, gastric cancer	[91, 98, 99]
MUC4	Salivary glands, airways, oesophagus, colorectum, female reproductive tract,	[80–82, 85, 94, 100]	Lung, ovarian, pancreatic, prostate, breast, gall bladder cancer	[16, 87, 93, 101– 103]
MUC5AC	Airways, stomach, pancreas	[16, 80, 83]	Breast, gastric, pancreatic cancer	[91, 93, 104]
MUC5B	Salivary glands, airways, oesophagus, pancreas, female reproductive tract	[16, 80–83, 100, 105]	Breast cancer	[93, 106]
MUC6	Stomach, pancreas, duodenum	[16, 83, 84]	Colon, gastric cancer	[91, 93, 107]
MUC7	Salivary glands, airways	[108, 109]	Gastric, colon cancer	[110] [111]
MUC8	Airways, male reproductive system	[109, 112]	Endometrial, cervical cancer	[113]
MUC9	Female reproductive system	[114]	Colon, ovarian cancer	[111, 115]
MUC11	Airways, colon	[80, 116, 117]	Colon	[111, 117]
MUC12	Pancreas	[16]	Colon	[117]
MUC13	Airways	[80]	Colon	[118]
MUC15	Placenta	[119]	Colon	[120]
MUC16	Ocular surface, Cornea, airways, female reproductive tract	[121–123]	Breast, Ovarian	[93, 123]
MUC17	Duodenum	[124]	Colon	[125]
MUC19	Airways, Salivary glands	[126]	Salivary gland cancer	[127]
MUC20	Airways, oesophagus, stomach, duodenum, colon	[109, 109, 128]	Salivary gland cancer	[109, 129]
MUC21	Airways, thymus, large intestine male reproductive system	[130]	Lung cancer	[130]
MUCL1	-		Breast	[131, 132]

In recent years functionally active TLRs have been identified in several cancers (table 4) [133, 134]. While several endogenous TLR ligands are proposed but, the molecular mechanisms of their action are currently a subject of debate [48]. It will be of interest to identify the detailed mechanisms of TLR activation in tumor cells, nevertheless the implications of constitutive TLR signaling are far reaching. Activation of pro-survival NF-κB pathway leads to an increased proliferation and chemoresistance [135, 136]. Several pro-inflammatory cytokines and chemokines such as TNFα, G-CSF, IL-1, IL-6, IL-8, COX-2, MCP-1 makes the microenvironment more conducive for the progression of cancer [137]. Immunosuppressive cytokines such as IL-10 and TGF-β dampen the immune response against growing tumor [137]. VEGF helps in angiogenesis nourishing the growing tumor. TLR signaling also helps in propagating tumor cells to distant sites by regulating cytoskeletal proteins and MMPs [134]. Interestingly, in some circumstances TLR signaling may inhibit the progression of cancer [137]. It is shown that polyI:C triggers cell death in several cancers [138–143] acting via TLR3 which might be due to its unique signaling by TRIF dependent pathway. Thus it is extremely important to carry out a systemic characterization of TLR signaling pathways to ensure that the therapeutic targeting is safe. The detrimental stimulation of TLRs on tumor cells can be avoided by selecting ligands which are recognized by immune cells present in the tumor microenvironment and not by cancer cell thereby preventing activation of the pro-survival TLR signaling pathways in cancer cells. Many TLR agonists and antagonists are in clinical trials for cancer as immunotherapeutic or chemotherapeutic agents [137, 144, 145]. Given the clinical relevance of TLRs, an elucidation of the role of TLR signaling in probable regulation of expression of MUCs will be highly useful. In the following sections we attempt to identify the probable mechanisms of regulation of MUC and TLR genes as illustrated in Figure 2. Identification of molecular mechanisms of interaction between MUCs and TLRs may identify new drug targets and strengthen our understanding on the mode of action of existing drugs.

Table 4.

The expression of TLRs in various human tumor tissues/cell lines and the techniques to study them – Reverse transcriptase PCR (PCR), Functional tests (FT), Western blot (WB), Flow cytometry (FC), immunofluorescence (IF), in-situ hybridization (ISH)

TLR	Type of Cancer/cell-lines/tissues	Technique used for the study	References
TLR1	colon	PCR	[146]
TLR2	colon	PCR,FT	[146, 147]
	melanoma	PCR,FC	[148]
	breast	PCR,WB,FT,FC	[149]
	hepatocellular	PCR,FT	[150]
	laryngeal	IHC	[151]
	ovarian	IHC,FT	[152]
TLR3	colon	PCR	[146]
	breast	IHC,WB	[153, 154]
	ovarian	IHC,FT	[152]
	lung	PCR,IF,FT	[155]
	melanoma	WB	[148]
	laryngeal	IHC	[151]
	prostate	IHC,PCR,WB	[156, 157]
TLR4	gastric	IHC	[158]
	colon	PCR	[146]
	ovarian	IHC,FT	[152]
	lung	PCR,FT	[159]
	melanoma	PCR,WB	[148]
	brain	PCR,FT,FC	[160]
	breast	PCR,WB,FT,FC	[161]
	laryngeal	IHC	[151]
	prostate	IHC,FT	[157, 162]
TLR5	gastric	IHC	[158]
	ovarian	IHC,FT	[152]
	cervical	IHC,FT	[163]
TLR9	gastric	IHC	[158]
	colon	PCR,FT	[164]
	cervical	IHC,PCR	[165]
	lung	IHC, ISH, PCR,IF,FT	[166]
	prostate	IHC,WB, FT	[157, 167]
	breast	DNA arrays, WB, IHC, FT	[168]
	hepatocellular	IHC, WB,FT	[169]

Fig. 2.

The proposed mechanisms of interaction between TLRs and MUCs: I. Ligand-TLR interaction initiates signaling pathways leading to the induction of several pro-inflammatory cytokines MAPKs and NF-κB which are known to induce mucin gene expression. Alternatively these molecules can also induce TLR expression in turn regulating mucin expression thereby forming a self-perpetuating signaling loop. Constitutively active TLR pathways are commonly observed in several cancers.

II. Mucins can also activate signaling pathways such as their interaction with receptor tyrosine kinase ErbB2 and other molecules activates signaling pathways which have common downstream signaling components and lead to a cross-talk with TLR signaling pathway. III. Mucins are large glycoprotein molecules which exhibit the receptor masking and can provide a steric hindrance for endogenous ligand binding to TLRs thus inhibiting their signaling pathway. IV. The sequestration of TLR ligands in the dense mucin matrix can have two possible outcomes either activation or inhibition of TLR signaling pathway. V. Altered glycosylation of mucin molecules can act as a ligand for TLRs and activate TLR signaling pathway.

5.1 Cross-regulation of MUC and TLRs in cancer

5.1.1 TLRs regulating MUCs expression

Inflammation is increasingly being recognized as the driving element for carcinogenesis [170, 171]. In addition to infection, inflammation links MUCs and TLRs in conditions such as inflammatory bowel disease [172, 173], colitis [174] and pancreatitis [173, 175] which are recognized as important risk factor for the development of cancer. Constitutively active TLR signaling in cancer cells leads to a state of ‘sterile’ inflammation [176]. Binding of ligands to TLRs initiate a complex signaling cascade leading to the activation of several pro-inflammatory cytokines, which via autocrine and paracrine effect initiate a cascade of intracellular signaling, altering number of cellular functions [9, 28]. Therefore, it is important to study the effect of cytokines from TLR signaling on MUC expression. Several pro-inflammatory cytokines are known to regulate the expression of different MUCs in cancer. The effect of IFN-γ, IL-1α and TNF-α was studied on the expression and glycosylation of MUC1, MUC5AC, MUC4 and MUC16 in multiple pancreatic cancer cell lines [7]. In bronchial epithelial cells IL-1β and IL-17A are potent inducers of MUC5AC synthesis [5]. MUC5AC induction by these cytokines was observed to be both time- and dose-dependent [5]. TNF-α, IL-1β and IL-6 induce MUC2 and MUC4 intestinal MUCs, which are detected in gastric tumor [177]. TNF-α and IFN-γ induce MUC1 expression in ocular surface epithelial cells [4]. TLRs signaling activate NF-κB and MAP kinases which are known to regulate the expression of different MUCs [6]. Thus activation of various signaling molecules, such as IRF3, NF-κB, ERK, p38 and JNK pathways by TLRs either directly or via inflammatory molecules might regulate MUC expression. Although not much is known it will be of further interest to see the role of chemokines, redox molecules and others mediators of inflammation on MUC expression.

5.2 MUCs regulating TLR signaling

MUCs interacting proteins

A series of studies have demonstrated a surprisingly diverse role for the small, highly conserved cytoplasmic domain of MUC1 in intracellular signaling. The cytoplasmic domain of MUC1 mucin is shown to interact with a large number of intracellular signaling molecules [178]. The cytoplasmic tail of MUC1 is 72-amino acid and has multiple phosphorylation sites which are responsible for interaction with several oncogenic proteins and their signaling [179]. For instance MUC1 contains multiple binding motifs for the proteins GSK3β, Src, β-Catenin and members of ErbB family [178] which may further interact and/or activate TRAF-6, NF-κB and MAP kinases and may participate in the expression of TLRs [180–187]. Thus, the cytoplasmic domain of MUCs can modulate TLR signaling and expression with one or more of these common signaling molecules. Unfortunately, limited information exists on the role of cytoplasmic domains of other MUCs. In addition to regulation of expression; MUCs can modulate TLR signaling pathways by multiple ways.

MUCs masking TLRs and ligand sequestration

The presence of large mucin molecules interfere with the ability of ligands to bind to the cell surface receptors principally by two methods [3]. It has been shown that the mucous layer captures and holds the biologically active molecules as a part of the matrix. In normal epithelia, large mucin glycoprotein molecules act as a molecular shield preventing the binding and attachment of ligands to the receptors. The extracellular domain of MUCs forms a matrix which masks the receptors and makes it inaccessible for binding with the ligands. As several tumors exhibit consistently higher expression of TLRs their masking by large mucin glycoproteins may actually affect the signaling pathways activated by them. The presence of large number of endogenous TLR ligands in the tumor microenvironment and their sequestration by MUCs might ultimately affect the fate of the tumor cells. As discussed previously MUC1 acts as a negative regulator of TLR5 signaling by binding with Pseudomonas aeruginosa flagellin [74]. In cancer, several molecules in the tumor microenvironment might get sequestered in the mucin matrix and may be unable to bind to the receptors on the cell surface. Alternatively, due to an increase in local concentration ligands may bind more efficiently and consistently to activate signaling through TLRs [3].

Altered glycosylation on MUCs acting as an intramembrane ligand for TLRs

TLRs sense an extremely diverse repertoire of ligands which range from exogenous microbial derived products to several host-derived endogenous molecules. In light of this diversity of ligand recognition by TLRs, there exists a possibility that altered glycosylated moiety of MUC molecules on tumor cells might act directly as TLR ligand thereby modulating the expression and signaling of these receptors. C-type lectin, a type of PRR, recognizes altered glycosylation on tumor cells and cross-talk with the TLRs pathway [188]. However, there is no report of TLR activation by altered glycosylation of self-molecules on tumor cells.

6.0 Conclusion and future directions

It is clear from the above account that MUCs and TLRs coordinate to mount an effective immune response against pathogens. Individually, MUCs and TLRs have both been found to be well associated with cancer. It will be important to study the interaction between the two in various disease conditions. Remarkably as MUCs generally appears since the initiation of cancer, a therapeutic intervention by TLR agonist/antagonists will be highly significant for early therapeutic intervention. An understanding of the molecular mechanisms of cross-regulation between MUCs and TLRs will be helpful in understanding the molecular mechanisms of TLR directed therapies. The proposition of altering MUCs expression by TLRs therefore holds great promise for translation from bench to bedside. Even after several decades of exhaustive research in the field, the current treatment strategy for cancer still remains inadequate. An understanding of the molecular mechanisms leading to disease development will certainly help in improving the efficacy of anti-cancer therapies.

Abbreviations used

TLRs

Toll-like receptors

MUC

Mucin

PAMP

Pathogen associated molecular pattern

VNTR

Variable number tandem repeat

TIR

Toll/IL-1 receptor

PRR

Pathogen recognition receptor

TFF

trefoil factor

LRR

Leucin rich repeat

NOD

Nucleotide oligomerization domain

LPS

Lipopolysaccharides

TRIF

TIR-domain-containing adapter-inducing interferon-β

MyD88

Myeloid differentiation primary response gene (88)

HMG

High-mobility group

HSP

Heat shock protein

IRAK

Interleukin-1 receptor-associated kinase

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

TNF-α

Tumor necrosis factor-alpha

IL

Interleukin

VEGF

Vascular endothelial growth factor

G-CSF

Granulocyte colony-stimulating factor

CCL

CC chemokine ligand

CXCL

CXC chemokine ligand

COX-2

Cyclooxygenase-2

TGF-β

Transforming growth factor beta

CXCR

CXC chemokine receptor

ICAM-1

Inter-cellular adhesion molecule-1

MAPK

Mitogen-activated protein kinase

JNK

c-Jun N-terminal kinases

ERK

Extracellular-signal-regulated kinase

IRF

Interferon regulatory factor

TRAF

TNF receptor associated factor

TAK-1

TGF-beta activated kinase 1

EGF

Epidermal growth factor

CA 15-3

Carcinoma Antigen 15-3

CA125

Cancer antigen 125 or Carbohydrate antigen 125

MCP-1

Monocyte chemoattractant protein 1

MMP

Matrix metalloproteinase

IFN-γ

Interferon-gamma

GSK 3

Glycogen synthase kinase 3

Src

Sacroma