Current status of mucins in the diagnosis and therapy of cancer

Abstract

Mucins are the most abundant high molecular weight glycoproteins in mucus. Their nature and glycosylation content dictates the biochemical and biophysical properties of viscoelastic secretions, pointing out an important role in diverse biological functions, such as differentiation, cell adhesions, immune responses, and cell signaling. Mucins are expressed in tubular organs by specialized epithelial cells in the body. Their aberrant expression is well documented in a variety of inflammatory or malignant diseases. From a prognosis point of view, their expression and alterations in glycosylation are associated with the development and progression of malignant diseases. Therefore, mucins can be used as valuable markers to distinguish between normal and disease conditions. Indeed, this alteration in glycosylation patterns generates several epitopes in the oligosaccharide side chains that can be used as diagnostic and/or prognostic markers. Furthermore, these characteristic tumor-associated epitopes are extensively used as appropriate immunotargets of malignant epithelial cells. Therefore, in an effort to detect and treat cancer at the earliest stage possible, mucins are analyzed as potential markers of disease for diagnosis, progression, and for therapeutic purposes. In this review, we focused on the current status of the distribution of mucins in normal and pathologic conditions and their clinical use both in cancer diagnosis and therapeutics treatments.

1. Introduction

Mucus is the slimy and viscoelastic secretion that covers the epithelial surface of tubular organs such as tracheobronchial, gastrointestinal, reproductive tracts, and other specialized organs. In the body, mucus is secreted by specialized epithelial cells known as goblet cells and are abundant in the epithelium of the gastrointestinal, respiratory and reproductive tracts, and the secretory epithelial surfaces of the liver, pancreas, gall bladder, kidney, salivary, and lacrimal glands [1]. Mucus secretions adhere to the epithelial surface and serve as a protective diffusion barrier against harmful substances and act as a lubricant between the lumen and the cell surface [2,3]. The composition of mucus varies with its location and pathophysiological conditions [4,5], but normally mucus is composed of water, inorganic salts, immunoglobulins, secreted proteins, and mucins. Mucins are the most abundant macromolecules in mucus and are responsible for its biochemical and biophysical properties due to their nature and extent of glycosylation [6,7]. The mucins are a closely related family of O-glycoproteins that play an important role in the renewal and differentiation of the epithelium, cell adhesions, immune response, and cell signaling [3,8–11].

In general, mucins are large (well over 10⁶ Daltons) glycoproteins [12,13] composed of ∼75% carbohydrate and 25% amino acids linked via O-glycosidic bonds between N-acetylgalactosamine and serine/threonine/proline (Ser-Thr-Pro) residues. The hallmark of the mucin family is the large and polymorphic central domain, which is composed of a variable number of tandem repeats (VNTR) rich in Ser-Thr-Pro residues (Table 1) that can be modified with a large number of O-linked oligosaccharides and a few N-glycan chains [3,6,7,14–16]. Till now, about twenty mucin (MUC) genes have been identified and these are designated as MUC1-2, MUC3A, MUC3B, MUC4, MUC5B, MUC5AC, MUC6-9, MUC11-13, MUC15-17, and MUC19-21 [3,17–31]. In this review, we discuss the current status of mucins for cancer diagnosis and therapy. Special emphasis is given on the most commonly occurring lethal cancers.

Table 1. Human mucins and their chromosome localization, domain structures.

MUCIN GENE	Chromosome location	Domains	AA in tandem repeat region
Soluble/gel-forming
MUC2	11p15.5	STP, VWC, VWD, CysD, and CK	50–100 TR of 23 AA
MUC5AC	11p15.5	STP, VWD, VWC, CysD, and CK	(TR1) 124/(TR2) 17/(TR3)34 and (TR4)66 TR of 8 AA
MUC5B	11p15.5	STP, VWD, CysD, and CK	11 TR of the irregular repeat of 29 AA
MUC6	11p15.5	STP, VWD, and CK	169 AA
MUC19	12q12	STP, VWD, VWC, and CK	3–6 TR of 19 AA
Soluble
MUC7	4q13.3	STP	6 TR of 23 AA
MUC8	12q24.3	STP incomplete sequence	13/41 AA
MUC9		STP	15 AA
MUC20	3q29	STP	3 TR of 19 AA
Membrane bound
MUC1	1q21-24	STP AND SEA, TM	25–125 TR of 20 AA
MUC3A	7q22	STP, SEA, EGF, and TM	20 TR of 17 AA
MUC3B	7q22	STP, SEA, EGF, and TM	17 AA
MUC4	3q29	STP, NTDO, AMOP, VWD, EGF, and TM	145–395 TR of 16 AA
MUC11	7q22	STP, SEA, EGF, and TM	28 AA
MUC12	7q22	STP, SEA, EGF, and TM	28 AA
MUC13	3q13.3	STP, SEA, EGF, and TM	8 TR of 27 AA
MUC15	11p14.3	STP and TM	No TR region
MUC16	19p13.2	STP, SEA, and TM	60 TR of 156 AA
MUC17	7q22	STP, SEA, EGF, and TM	61 TR of 59 AA
MUC21	6	STP and TM	28 TR of 15 AA

STP, domain rich in serine, threonine and proline, which are heavily glycosylated; VWD von Willebrand factor type D domain; VWC, von Willebrand factor type C domain; CK, Cys-knot domain; SEA, Sea urchin sperm protein, Enterokinase, and Agrin module (MUC16 has six SEA domains—five of them in the tandem repeat region); NIDO, Nidogen-like domain; EGF, epidermal growth factor-like domain (MUC3A. 3B. 4, and 12 have two EGF-like domains and MUC13 has three); AMOP, Adhesion-associated domain in MUC4 and other proteins; TM, transmembrane domain; TR, Tandem repeat; AA, Amino acid.

2. Classification of mucins

Based on physiological fate and nature, mucins are categorized into three subgroups: “secreted/gel-forming”, “membrane-bound”, and “soluble” mucins [3] (Table 1, Fig. 1). The first group is composed of strictly secreted, gel-forming mucins including MUC2, MUC5AC, MUC5B, MUC6, and MUC19, which form oligomeric structures. The second group is composed of mucins either tethered at the cell surface or secreted in the mucus. The mucins of this group, MUC1, MUC3A, MUC3B, MUC4, MUC11, MUC12, MUC13, MUC15, MUC16, MUC17, MUC20, and MUC21, harbor a transmembrane domain, a short cytoplasmic tail (CT), and an extensive extracellular domain. The third subgroup, composed of MUC7, MUC8, and MUC9, are exclusively secreted non-gel forming mucins.

Fig. 1.

A schematic representation of the deduced amino acid for various MUC genes. SEA, Sea-urchin sperm Protein; Ig, Immunoglobulin; pVW, pro-Von Willebrand; AMOP, Adhesion Associated Domain; NIDO, Nidogen-like Domain; EGF, Epidermal Growth Factor; TM, Transmembrane.

2.1. Secretory/gel-forming mucins

Out of the gel-forming mucin family, MUC2, MUC5AC, MUC5B, and MUC6 genes are mapped to chromosome 11p15.5, in a cluster of 400 kb in size, rich in CpG islands [32]. These are localized between HRAS and IGF2 and arise from a common ancestor gene similar to the human von Willebrand factor gene (vWF) [33,34]. On the other hand, MUC19 is localized to chromosome 12q12. This family of mucins is formed by oligomerization of the tri-dimensional network of the mucus backbone, which provides the mucus its visco-elastic properties. The gel-forming mucins share several structural features including a large central repetitive region flanked by a non-repeating region. The central region is comprised of tandem repeats varying in length from 24 nt for MUC5AC [35] to 507 nt for MUC6 [27]. The tandem repeat domains encompassed several sub-domains rich in cysteine residues. The number of these sub-domains varies depending of the mucin, which allows mucin dimerization. The amino and carboxy-flanking regions share similarities with the B, C, D, and CK domains found in the pro-von Willebrand factor (pro-vWF) [19–21,23,35–37].

MUC2 was first identified and described by Gum et al in 1989 [38]. Its cDNA was isolated from a human small intestine cDNA library [23,38]. The central domain of MUC2 is composed of two highly repetitive sequences. The first, in the central position, is characterized by the perfect repetition of one motif of 23 amino acids and the second, located upstream, is composed of an irregular sequence repeated in tandem with a unit of 347 amino acids. These two sequences are rich in amino acid residues of Ser-Thr-Pro. MUC2 is mainly expressed in intestinal [39] and colorectal goblet cells [38,40,41]. The pathogenesis of colorectal neoplasia is associated with MUC2 expression [42]. Downregulation of MUC2 expression is detected in colorectal cancer [42], gastric cancer [43,44], and ovarian tumors [45]. It is also expressed by adenomas and mucinous carcinomas [42].

The MUC5AC is primarily expressed in tracheobronchial goblet cells, in gastric epithelial cells [46,47], in conjunctiva and lacrimal gland tissues, but not in cornea [48]. The abnormal expression of MUC5AC has been observed in colorectal adenomas [49,50] and pancreatic cancer [51,52]. Furthermore, the expression level of MUC5AC is associated with the degree of cellular dysplasia. It is also expressed at higher level in patients with asthma, bronchitis, cystic fibrosis [53,54], and Sjögren syndrome. Recently published work states that MUC5AC is more frequently expressed in advanced cholangiocarcinoma [55]. In contrast, a decrease in MUC5AC expression is associated with decreased survival in colorectal carcinoma patients [56,57].

MUC5B is the second largest human gel-forming mucin, composed of 5,000 amino acids [19,20]. It is detected in human saliva, salivary glands, tracheobronchial and esophageal epithelia, pancreatobiliary and endocervical epithelial cells [46,47,58]. Both MUC5B and MUC5AC mucin proteins have been identified in human airway secretions from healthy and diseased individuals [59–63]. MUC5AC and 5B expression is decreased in cystic fibrosis airway secretions [64].

MUC6 is expressed in gastric mucosa and mucopeptic cells located at the neck region of the body [65–69]. It is also expressed in gastric and duodenal mucous glands, pancreatobiliary and endocervical epithelial cells [69], and in the stomach of 8-week-old embryos [70]. The aberrant expression of MUC6 is reported in gastric carcinomas. In addition, MUC6 expression is detected at significant levels in well-differentiated cholangiocarcinomas [55], breast cancer [66], and in colonic sessile serrated adenoma [71].

The human MUC19 gene is the largest gel-forming mucin with a size of 180 kb and with a deduced peptidic sequence of more than 7,000 amino acids. It is localized at chromosome 12q12 [72]. In the human middle ear, it is the major gel-forming mucin [73]. It is also expressed in mucous cells of sublingual and submandibular glands, submucosal gland of the tracheal tissue [72] corneal and conjunctival epithelial cells, and lacrimal gland tissues [48]. However MUC19 is not detected in salivary glands [74] and its expression is decreased in conjunctiva of Sjögren syndrome patients.

2.2. Soluble mucins

Originally, the MUC7 cDNA clone was isolated from the human submandibular glands (Reddy et al., 1992), and later on, the full-length cDNA was isolated and characterized from the same cDNA library [17]. It is a gene of 10 kb long, localized to chromosome 4q13.3 and composed of three exons only. It is a low-molecular mass mucin (150–200 kDa) and is secreted by the submandibular and sublingual salivary glands. It encodes a 39 kDa protein of 377 amino acid residues and reveals five distinct domains [75]. It functions as an antimicrobial agent in the oral cavity by interacting with microorganisms. It also agglutinate the AIDS virus HIV-1 [76,77] and inhibits the HIV infection [78]. This is mainly due to the two cysteine residues located in the N-terminal region of MUC7 [79]. Moreover, it acts as an anti-candidal activity [80] via its histatin-like domain. The MUC7 is expressed in epithelial cells of the oral cavity, minor salivary gland, respiratory tract [81], normal human conjunctivae [82], pancreas, submucosal glands of the bronchus, and cholangiocarcinomas [83–85]. The MUC7 gene is also expressed in bladder cancer during the malignant transformation of bladder urothelium in preinvasive carcinoma in situ but no MUC7 gene expression is detected in superficial, non-invasive bladder tumors [86].

The MUC8 mucin gene has been mapped to chromosome 12 at position q24.3. The MUC8 gene mRNA and protein is expressed in nasal epithelial (NHNE) cells [87], in middle ear epithelium [88], and in middle ear effusions [89]. The expression of MUC8 is up-regulated in nasal polyp epithelium and it may play an important role in the pathogenesis of chronic sinusitis with polyps [90].

The expression of MUC9 was observed from late follicular development through early cleavage-stage in embryonic development, middle ear epithelium [88], and middle ear effusions [89].

2.3. Transmembrane mucins

Membrane bound mucins are tethered to cells with the help of a unique transmembrane domain. The mucin cytoplasmic tails facilitate their association with cytoskeletal elements and cytosolic adaptor proteins, and therefore, these have a significant role in signal transduction [91]. The family of membrane-associated mucins has several common features such as a specific proteolytic cleavage site, two or three epidermal growth factor (EGF) domains, juxtamembrane domain, tandem repeat, and a cytoplasmic tail. In MUC3A, MUC12, MUC13, and MUC17, the EGF domains are separated by the sea urchin sperm protein (SEA) domain, but not in MUC4.

In 1990, MUC1 was sequenced at the DNA level by four groups [25,92–94]. Subsequently, it was mapped to chromosome 1q21-24, spanning about 4–7 kb of genomic DNA, depending upon the size of the central domain [95–98]. MUC1 consists of seven exons, out of which, exon 1 encodes the leader peptide, exon 2 the central domain, and exon 6 and 7 encode the transmembrane sequence and cytoplasmic tail. The central domain is made up of a sequence repeated in tandem with a perfect unit repetition of 20 amino acid residues [99]. MUC1 was first identified in human milk [100] and is expressed by almost all glandular epithelial surfaces of respiratory, female reproductive, gastrointestinal tracts, middle ear, salivary gland, mammary gland [100–104], and normal pancreatic intralobular ducts [105,106]. Its expression is dramatically increased in breast, ovarian, lung, pancreatic, prostate [85,107,108], and colorectal cancers [109,110]. In secretory epithelial cells, MUC1 is normally expressed on the apical borders, but in tumor cells, its expression is spread throughout the cell surface [100,111]. One major role of the MUC1 protein is to act as an anti-adhesive protein to maintain the luminal integrity of the lining epithelium, thus aiding in the metastasis of tumor cells [112,113].

MUC3A and MUC3B are expressed in gastrointestinal epithelia [114]. The MUC3 gene has been mapped to chromosome 7 in the region q22 [22,115]. It is a large gene, and the sequence is partially known as the organization of its central domain is unclear. The alternative splicing results in four variants which may be membrane bound or secretory [28,116].

The first MUC4 mucin sequence was cloned from a tracheobronchial cDNA library [29] and, subsequently from colon mucosa [117] and pancreatic tumor cell lines [118]. It has been mapped to the chromosome 3q29 [119] close to the small membrane bound mucin MUC20 [120]. MUC4 is made up of 26 exons, in which, the largest one being exon 2 codes for a large central tandem repeat domain rich in Ser/Thr/Pro [18,121]. The first exon covers the 5′ untranslated region and codes for the leader peptide, whereas the remaining 24 exons code for the extracellular, transmembrane, and carboxyl tail of the MUC4. This glycoprotein is expressed in various normal tissues such as the respiratory tract, lung, salivary glands, stomach, colon, eye, vagina, ectocervix, uterus, and prostate [15,46,122–127]. It is not detected in the normal pancreas, gall bladder, liver, and biliary epithelial cells [47,51,70]. Additionally, an abnormal expression of MUC4 has been observed in various inflammatory diseases and cancers such as dysplasia and adenocarcinoma of the esophagus [128], Crohn's disease [129,130], gall bladder carcinomas [131], salivary gland mucoepidermoid carcinoma [132], lung adenocarcinoma [133–135], pancreatic ductal adenocarcinoma [51,136–138], epithelial ovarian carcinomas [139], prostate cancer [140], breast cancer [141], and other inflammatory diseases of airways such as cystic fibrosis (CF) and chronic obstructive pulmonary disease [5,142].

The MUC11 mucin gene was mapped to the human chromosome 7q22 region [143], similar to MUC12 and MUC3. Up to date, only a partial sequence of MUC11 has been isolated, corresponding to a part of the tandem repeat domain. This sequence presents 71% homology with the variable repeat region of MUC12. At the RNA level, the expression of MUC11 was highest in the colon, whereas, a weak expression was observed in the stomach, pancreas, prostate, and uterus [114,143]. The MUC11 is expressed more widely in gastrointestinal, respiratory, reproductive, urinary tracts and also detected in the liver and thymus when compared with MUC12 and MUC3. Its expression is commonly down-regulated in colorectal cancer [143].

The MUC12 mucin gene has been mapped to the human 7q22 region [143]. It spans at least 28 kb long and likely contains 13 exons. It has structural similarities with MUC3, with two EGF-like domains, one transmembrane sequence, and a cytoplasmic tail. MUC12 is most abundantly expressed in the normal colon and stomach, and to a lesser extent in the pancreas [114,144], prostate, and uterus.

The MUC13 gene was mapped to chromosome band 3q13.3 [145]. It encodes a cell surface membrane-anchored mucin expressed in the normal gastrointestinal, respiratory tracts, trachea, middle ear, and kidney [88,114,145,146]. It is aberrantly expressed in colorectal, esophageal, gastric, pancreatic, and lung cancers [144,146]. In colorectal cancers, the highest expression of MUC13 was observed in poorly differentiated tumors. The staining intensity was high in adenocarcinomas (81%) when compared with mucinous tumors (50%). Similarly, the high-intensity of cytoplasmic staining was observed in poorly differentiated and late-stage tumors [146].

The human MUC15 is localized to chromosome 11p14.3 and contains five exons and four introns. It is made of a signal peptide and the majority of the extracellular fraction is encoded by exon 3, which is followed by a 150-bp exon encoding the transmembrane domain (exon 4). The cytoplasmic domain is encoded by exons 4 and 5, which also contains a 274-bp-long 3′ untranslated region and the stop-codon. The MUC15 expression was abundant in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and also in leukemia K-562 cell line, but moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph nodes, adult and fetal thymus [147,148]. Abnormal expression of MUC15 has been observed in (mRNA and protein) colorectal tumors when compared with the normal tissues [149] and its expression enhances the invasive potential of colorectal cells.

The MUC16 encodes a transmembrane-bound molecule also known as CA125. It was cloned from the ovarian cancer cell line OVCAR-3 and was mapped to chromosome 19p13.2 [122]. It encodes splice variants that give rise to polypeptides that differ in the number of SEA domains repeated 7, 12, or 60 times [150]. The first MUC16 cDNA clones were published by two independent groups [151,152]. The MUC16 translated into a large peptide made up of 22,152 amino acids with a molecular weight of 2.5 MDa. It has a large extracellular domain extending up to 500 nm from the cell surface into the glycocalyx, a transmembrane domain, and a small cytoplasmic tail. The extracellular domain is composed of a heavily O-glycosylated N-terminus, a tandem repeat region, and a region with several SEA domains near the membrane-spanning sequence. MUC16 is mainly expressed in the ocular surface, respiratory tract, and female reproductive tract epithelia [153], and middle ear [88]. Its overexpression is correlated with the progression of ovarian cancer [154].

The MUC17 gene was mapped to the mucin cluster at the chromosomal locus 7q22 along with MUC3A/B, MUC11, and MUC12 mucins [155,156]. The first partial length cDNA sequence of MUC17 was identified by Van Klinken and co-workers [157]. MUC17 was extended to its 5′-part using genomic sequences by 5′RACE-PCR [158]. It is made of 13 exons, out of which, the exon 3 is the largest one (12.2 kb) and codes for the Ser/Thr/Pro region. This domain is followed by two EGF modules flanking a SEA domain, a transmembrane (22-amino acid) domain, and an intracytoplasmic tail (78-amino acid). The MUC17 is expressed in the gastrointestinal tract showing the highest expression in the duodenum and transverse colon [156,159]. The MUC17 is also expressed in the stomach, fetal kidney, and conjuctival epithelium [156,160]. The MUC17 is overexpressed in pancreatic cancer cells when compared with the normal pancreas and pancreatitis tissues [138].

MUC20 has been localized to chromosome 3q29 and contains five exons coding for a 76–78 kDa protein. Abundant expression of MUC20 mRNA was detected in the kidney but moderately in placenta, lung, prostate, liver, colon, esophagus, rectum [120], and middle ear [88]. The expression of MUC20 is up-regulated in immunoglobulin A nephropathy (IgAN) [120,161].

The epiglycanin/MUC21 gene was mapped on chromosome 6 in close proximity to the MHC class I and within the susceptibility domain for diffuse panbronchiolitis (DPB) [162]. It spans 4.8 kbp in size and consists of three exons coding a 21 amino acid signal peptide, the tandem repeat domain, the transmembrane domain, and the cytoplasmic tail. It encodes a typical transmembrane mucin with a cytoplasmic tail, a transmembrane domain, a stem domain, and 28 tandem repeats composed of 15 amino acids. Human epiglycanin/MUC21 mRNA expression was examined in various normal tissues by PCR screening using human epiglycanin/MUC21 specific primers. It was demonstrated that expression was remarkably high in lung, thymus, colon, normal bronchi, bronchioles, and bronchial glands, large intestine, and testis [163].

3. Mucins as diagnostic markers for various cancers

Worldwide, mucins are extensively studied for their clinical use as carcinoma markers and for cancer immunotherapy [2,164]. Tumors associated epitopes are generated in the oligosaccharide side chains of mucins due to changes in their glycosylation patterns, including newly, expressed blood-group antigens. Therefore, in an effort to detect cancer at the earliest stage, mucins are analyzed as potential markers of disease progression and diagnosis. Significant findings are discussed below and are subdivided by the tissue where the primary tumor originated.

3.1. Breast cancer

MUC1 is overexpressed frequently in breast cancers and is a target for diagnostic assays and immunotherapy [165]. Fifteen years ago, a helper-T cell independent response directed to the PDTR region of unglycosylated MUC1 TR peptides was reported in the sera of breast, colon, and pancreatic cancer patients [166]. In these studies, IgM antibodies specific to MUC1 were detected using a synthetic 105-residue peptide that mimicked the native structure of unglycosylated MUC1. Humoral responses specific to MUC1 were detected in 8.3% of breast cancer, 16.7% of pancreatic cancer, and 10.0% of colon cancer patients without any correlation to tumor. Thus, as a diagnostic tool, this approach was of little value as fewer cases of the cancer patients studied had detectable circulating MUC1 levels.

In breast cancer patients, a shortened five-year disease-free interval, increasing tumor node metastases stages, positive lymph node status, and increasing histological grade are significantly associated with the expression of the mucin associated Tn antigen [167]. In agreement with these studies, a better overall five-year survival was associated with Sialyl-Tn negativity [168]. Further, the increased expression of Lewis x and sialyl-Lewis x correlated with lymph node metastasis and poorer survival [169,170]. A study that was conducted to compare the superiority of serum biomarker (CEA, CA15.3, and CA27.29) to diagnose patients with primary breast cancer showed that CA15.3 was superior to CA27.29 [171], while other studies reported no difference among these markers [172].

One recent study demonstrated the potential use of mucins as prognostic indicators in breast cancer patients [173]. The circulating tumor cells (CTC) in the blood of breast cancer patients undergoing palliative therapy were evaluated for the expression of MUC1 among other markers. CTC analyzed by immunomagnetic tumor cell selection and multiplex RT-PCR, were detected in 52% of the patients and 86% of this group was MUC1 positive. The presence of CTC correlated with shorter overall survival when compared with patients that did not have detectable CTC. Keeping in mind that CTC were selected by MUC1 specific expression, it can be inferred that MUC1 is a significant prognostic factor as well. The aberrant cytoplasmic and membranous localization of MUC1 in breast cancer tissues is associated with a poor outcome when compared with normal physiological apical localization, [141]. Recently, the MUC1 antibody named PankoMab, which binds specifically to carbohydrate-induced conformational tumor epitope on MUC1, revealed a strong correlation of MUC1 with hormone receptor expression in ductal carcinoma of the breast [174]. In the same study, PankoMab performance was compared with the VU-4-H5 antibody, which recognizes the tandem repeat region of MUC1, and an increased staining intensity was demonstrated in correlation with increased grading and lymph node involvement. Therefore, the combination of PankoMab and VU-4-H5 staining is suggested as a useful prognostic indicator of ductal breast cancer.

Another mucin, MUC3, was evaluated for breast cancer diagnosis. MUC3 membranous expression in breast cancer tissues is associated with poor prognostic outcome, higher tumor grade, and poorer Nottingham Prognostic Index (NPI) [141]. MUC3 expression was detected in 91% of human breast cancer tissues, and its expression associated with increased local recurrence and lymph node stage [141]. In other studies, the MUC5B was detected in primary breast tumors (81%), non-malignant breast diseases (92.8%), samples of normal breast epithelia adjacent to cancer (42.1%), and in none of the normal control breast epithelium [175].

3.2. Colon cancer

The tumor-associated glycoprotein (TAG-72) is aberrantly expressed in a variety of epithelial malignant tissues. The increased levels of serum TAG-72 antigen was observed in 48% of 56 gastric carcinoma patients and 67% of 45 colorectal carcinoma patients[176]. The B72.3 monoclonal antibody (MAb) detects an epitope carried by high-molecular-weight mucins (tumor-associated glycoprotein, TAG-72) recently identified as sialyl-Tn, and it has a restricted pattern of reactivity with normal tissues but it reacts with a high proportion of epithelial cancers. This antibody reacted strongly with 100% of the specimens of the mucosa adjacent to human colorectal adenocarcinomas (transitional mucosa) and with 81% of the specimens of adjacent colon cancer. Therefore, the expression of TAG-72 antigen occurs in the colon during the process of epithelial cell transformation [177].

The largest study evaluating the prognostic value of MUC1 and MUC3 expression in colorectal carcinoma samples was published in 2007 [178]. High throughput tissue microarray technology was used to evaluate 462 tumor samples. The study found that 32% of the tumors were positive to MUC1, and 74% were positive to MUC3. Although there was no correlation of both mucins in the clinicopathological variables, such as tumor stage and vascular invasion by univariate analysis, but multivariate analysis indicated that MUC1 expression was an independent marker of prognosis. Patients with higher MUC1 expression had a significant reduction in disease specific survival. Interestingly, the expression of MUC1 in colorectal carcinoma is a valuable indicator of poor prognosis in Caucasian patients when compared with those of African–American descent [179].

MUC2, MUC6, MUC12, and MUC13 expression was also investigated in colon cancer. Increased expression of MUC13 in colorectal cancers is associated with poorly differentiated tumors [146], whereas, MUC2 expression tends to be related to the indolent behavior of human neoplasms and a favorable outcome of the patients [180]. In the case of MUC6, it was found to have 100% specificity in distinguishing colonic sessile serrated adenoma (N = 26; positive staining) from hyperplastic polyp (N = 48; negative staining) [71]. In the case of MUC12 expression, this was down-regulated or absent in 6 out of 15 (40%) colorectal tumors samples, when compared with matched normal colonic tissues and the expression was down-regulated or not detected in any of six colorectal cancer cell lines examined in published work [155]. On the contrary, in colorectal cancers, the highest expression of MUC13 was observed in poorly differentiated tumors. The staining intensity was high in adenocarcinomas (81%) when compared with mucinous tumors (50%). Similarly, the high-intensity of cytoplasmic staining was observed in poorly differentiated and late-stage tumors [146].

3.3. Gastric cancer

In an attempt to evaluate whether membrane mucins or secretory mucins had a better prognosis value in gastric carcinoma, the expression of MUC1, MUC2, MUC3, MUC5AC, and MUC6 was evaluated in 46 tumor samples [181]. Normal gastric samples were positive to MUC1, MUC5AC, and MUC6. A shift in mucin expression was found on intestinal metaplasia, where an increase in MUC2 and MUC3 and a decrease in MUC1, MUC5AC, and MUC6 were obtained. Nevertheless, a drastic shift was not observed with gastric carcinoma samples as a measure of cancer progression. The main findings of this study was that a high expression of MUC1 correlated with a better prognosis, including smaller tumor size and an absence of metastasis, whereas MUC3 expression correlated with opposite trend. In addition, no prognostic correlation was found for the expression of secretory mucins and the development of gastric cancer.

In a recent published study, 133 mucinous gastric carcinomas (MGC) were compared with nonmucinous gastric carcinomas (NMGC) [182]. Key findings included that MGC tumor had deeper invasion, frequently metastasized to lymph nodes, and had a more advanced pathologic state than NMGC. In particular, MUC2 was expressed in 95.5% of MGC and in 33.4% of NMGC, suggesting a correlation between a mucinous histology and MUC2 expression in gastric carcinoma. However, the mucinous expression itself could not be identified as an independent prognostic factor of the progression of the disease. It appears that the aggressive characteristics of the MGC are a consequence of the advanced stage of the disease and the extracellular mucin facilitates the spreading of metastasis to the surrounding tissues.

In gastric cancer, MUC5AC expression was observed more frequently (100%) in lower grade carcinomas when compared to late stage carcinomas (58.6%) [68,183], and its expression is associated with better prognosis, whereas, its reduced expression is associated with shorter survival [184]. On the other hand, overexpression of MUC1 has been associated with poor clinicopathological parameters and poor survival [184]. The expression of sialyl-Lewis a [185] and Lewis x [186] epitopes indicates poor survival.

3.4. Lung cancer

Similar to other types of cancers, the MUC1 overexpression in squamous cell cancer of the lung is associated with poor survival [187]. Another mucin that has been envisioned as a potential candidate for tumor diagnosis is MUC4. Lung carcinoma tissues have been evaluated for the expression of MUC4 protein as well as mRNA levels [188]. Twenty-nine tumor samples were included in this study and it was found that 67% were positive for MUC4 protein expression and 72% were positive to MUC4 mRNA expression. Unfortunately, this expression could not be correlated to the clinical stage of cancer. Nevertheless, opposing to these results, a recent study demonstrated that the expression of MUC4 is in fact correlated with poor patient prognosis [189]. It was showed that patients, whose tumors had high MUC4 levels, had a disease-free curve significantly lower than in patients who had lower MUC4 levels [189]. Although only 13.5% of the patients included in the study had a high expression of MUC4, these results indicate that MUC4 expression is an indicator of poor prognosis in lung adenocarcinoma. Consistent with these studies, MUC4 overexpression in lung adenocarcinoma proved to be useful in distinguishing lung adenocarcinoma from malignant mesothelioma, with 91.4% sensitivity and 100% specificity [133].

3.5. Ovarian cancer

Ovarian cancer is another hidden disease that presents no detectable signs and hence eludes early detection. Recently, our group has published a comprehensive review of mucins in diagnosis, prognosis, and therapy of ovarian cancer [190]. From the diagnostic point of view, the concentration of MUC16 (CA125) increases in 80–90% of advanced stage ovarian cancers [191] and is the more widely used marker for its diagnosis [139,192]. Nevertheless, the detection of MUC16 as a diagnosis marker is limited due to its elevation in endometriosis, pelvic inflammatory disease, and pregnancy. Recently, Chauhan et al. showed that MUC4 (92%), MUC1 (83%), and MUC16 (79%) were significantly overexpressed in ovarian tumors and the expression of MUC4 was significantly higher when compared to MUC1 and MUC16 in early-stage ovarian tumors with 100% incidence. They suggested that, MUC4 could be a possible candidate biomarker for the early diagnosis of ovarian carcinoma and also indicated that MUC4 can be utilized in combination with MUC1 and MUC16 to achieve greater sensitivity for the detection of late-stage tumors [139]. Other studies that show contradictory results found no association between MUC4 expression and prognosis [193]. Other suggestions for an improved detection of stage 1 ovarian cancer include the combination of MUC16 (CA125), MUC1 (CA15-3), and tumor-associated glycoprotein 72 (CA72-4) [194].

3.6. Pancreatic cancer

To date, there is no specific tumor marker to diagnose pancreatic cancer, but mucins have been researched as potential candidates for this aggressive disease. Metzgar et al. (1982) raised monoclonal antibodies, such as DUPAN-2, specific to human pancreatic antigens (MUC1) secreted in the sera of pancreatic cancer patients [195]. Subsequently, this antibody was extensively studied in the United States [196,197] and in Japan [198] using a competitive assay and indicated a high positivity rate for DUPAN-2 in pancreatic and biliary tract carcinomas but low positivity in colorectal and gastric cancers. Later on, several researchers reported that mucin associated antigens like CA19.9, CA50, and CA195 [199–203] are elevated in advanced colorectal and pancreatic cancer and thus can be used as diagnostic markers.

The examination of MUC1 levels in serum was shown to be a good survival predictor for metastatic pancreatic cancer patients [204]. Similarly, determination of CA19-9 or sialylated Lewis antigen, along with ultrasound, computed tomography (CT) scan or endoscopic retrograde cholangiopancreatography (ERCP), can increase the positive predictive value from 62–71% to 100% [205]. In another study, the sensitivity of the CA19-9 serum assay ranged from 69 to 93% and the specificity varied between 46 and 98% [206]. The patients with invasive carcinoma of pancreatic and intrahepatic bile duct tumors showed poor outcome when the expression pattern was MUC1 positive and MUC2 negative, whereas, the non-invasive tumors with a favorable outcome exhibited an expression pattern of MUC1 negative and MUC2 positive [44,207]. Therefore, the expression pattern of MUC1 and MUC2 can be used as potential markers to differentiate between the invasive and non-invasive carcinomas of the pancreas and intrahepatic bile duct.

MUC4 is one of the candidate biomarkers in the diagnosis of pancreatic cancer [136,140]. It is not expressed in the normal pancreas or chronic pancreatitis, but it is highly expressed in human pancreatic tumors [51,136,208]. The MUC4 is expressed as early as in precancerous pancreatic intraepithelial neoplastic (PanIN) lesions, and its expression increases with the disease progression [137]. Studies have also shown that the detection of MUC4 is one of the useful markers for the early diagnosis of pancreatic cancer in fine-needle aspirates (FNA), exhibiting 91% sensitivity and 100% specificity [209]. It has also been reported that, MUC4 overexpression and a decreased expression of clusterin-β in pancreatic adenocarcinoma are useful in distinguishing reactive ductal cells from the cells of pancreatic adenocarcinoma in FNA samples.

Detection of circulating MUC1 by PAM4-based immunoassay could be useful in the diagnosis of pancreatic cancer [210]. In the same way, the quantitative assessments of mRNA, such as MUC1 and MUC5AC in pancreatic juice are potential candidate genes for preoperative diagnosis of pancreatic cancer [176]. A recent review described the presence of MUC4-specific antibody in the serum and MUC4 transcript in peripheral blood mononuclear cells (PBMCs) of cancer patients, positioning MUC4 as a promising diagnostic biomarker in high-risk individuals as well as in those with established cancer [211].

3.7. Prostate cancer

In clinical studies developed for targeted radioimmunotherapy in metastatic prostate cancer patients, the BrE3 antibody, which recognizes epitopes in the VNTR region of MUC1, showed a strong correlation of prostate cancer progression [212]. These results demonstrate the potential of hypoglycosylated MUC1 as a marker for aggressive prostate cancer.

3.8. Other cancers

The efficacy of mucins in diagnosis has been investigated in other less common cancers as well, and some of these examples are mentioned below, including bladder cancer, extrahepatic bile duct carcinoma, and cholangiocarcinomas. MUC2 expression is detected by the monoclonal antibody 4F1 in ∼40% of the cases of carcinomas of bladder, whereas normal urothelium showed no MUC2 expression [213]. In other study, the 111Indium-labeled anti-MUC1 mucin monoclonal antibody was detected both in primary and recurrent invasive bladder tumors with distant metastases. The use of this MUC1 radio labeled antibody will likely improve the clinical staging of cancers and assist in selecting radical therapy [53]. In extrahepatic bile duct carcinoma, MUC1/Df3 was the most useful indicator for prognosis among the various glycoforms of the MUC1 mucin [214]. Finally, the MUC1 expression in cholangiocarcinomas has been associated with dedifferentiation, infiltrative growth pattern, and patient survival, while an abnormal MUC1 expression is correlated with the progression of cholangiocarcinoma [55].

4. Evaluation of mucins in immunologic therapeutics

The use of mucins as targets for cancer therapy has been evaluated for the last 3 decades [2,140,215–225]. Altogether, most of the approaches involve instructing the immune system to recognize these glycoproteins presented in malignant tumor cells and eradicate them. A summary of the key findings in the immunotherapy of breast, colon, lung, pancreatic, prostate, and renal adenocarcinomas is presented below (Table 2).

Table 2. Mucins used as therapeutic targets in various cancers.

Primary cancer	Mucins in therapy	Therapy approaches	Major therapy outcomes
Breast	MUC1	TR peptides Alone Conjugated with KLH DC-based Vaccinia virus vectors	Stimulation CTL, NK responses Humoral responses
Colon	MUC1, Thomsen-Friedrich (T-Tn), Sialyl-Tn antigens	Modified desialylated mucin antigens plus adjuvants Antigens conjugated with KLH plus adjuvants Viral vectors expressing CEA and MUC1 plus costimulatory molecules	Humoral responses CTL responses T-helper responses
Lung	MUC1	Liposomal vaccine plus adjuvants and lipids	CTL responses
Ovarian	MUC1	TR peptides Tn-MUCl 90Y-HMFGl radio-labeled MUCl monoclonal antibody	CTL responses Humoral responses Median survival increased over 10 years
Pancreatic	MUC1	Vaccinia and fowlpox viral vectors plus costimulatory molecules and adjuvants	CTL responses
	MUC4	DC-based + T cell epitope PADRE	CTL responses
Prostate	MUC1	Vaccinia virus expressing MUC1 and IL-2 111In/90Y-ml70 radio-labeled MUC1 monoclonal antibody + paclitaxel	Disease progressed Metastatic cancer targeted Therapeutic index increased
Renal	MUC1	TR peptides	CTL responses

TR, Tandem Repeat; KLH, Key Limpet Hemocyanin; DC, Dendritic Cell; CTL, Cytotoxic T Lymphocytes; NK, Natural Killer Cells; CEA, Carcinoembryonic Antigen; PADRE, Pan-DR-epitope

4.1. Breast cancer

Breast cancer is the most investigated cancer for the development of mucin therapies. One of the earliest studies highlighting the immunogenicity of mucins in breast and pancreatic cancer dated from 2 decades ago [223]. Cytotoxic T lymphocytes (CTLs) were generated from lysed tumor cells collected from tumor draining lymph nodes of breast cancer patients in a MHC-unrestricted manner. The CTLs were cytotoxic to breast and pancreatic cancer cell lines and not to normal counterparts, implying that immunologic epitopes are not revealed in normal cells.

A clinical trial including 10 breast cancer patients that were immunized with MUC1 33-mer and 106-mer MUC1 tandem repeat peptide found high levels of circulating MUC1 antibodies after the immunizations [226]. MUC1 antibodies, present in the plasma of immunized patients, were cultured with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. It was demonstrated that tumor cell lysis specific to MUC1 was induced by the stimulated PBMCs. Natural killer cells were the essential effector cells for the MUC1 antibody-mediated cytotoxicity, indicating the involvement of both innate and adaptive immune systems.

In an effort to increase the immunogenicity of the MUC1 vaccine, the mucin has been conjugated with several proteins. A promising study with metastatic breast cancer patients that were immunized with a 16-mer MUC1 peptide (BP16) conjugated to the carrier protein keyhole limpet hemocyanin (KLH), shows that MHC I restricted CTLs precursors were generated in vitro with synthetic MUC1 peptide [227]. In another approach, in a phase I clinical trial involving 25 patients with metastatic adenocarcinoma of breast, colon, rectum, and stomach, strong humoral responses were generated when oxidized mannan-MUC1 fusion protein was injected in the patients [228]. Around 70% of the patients receiving eight injections yielded strong MUC1-antibodies responses, while CTL responses were present in only two out of 10 patients. In a recent published clinical study, the vaccine formulation also consisted of oxidized mannan-MUC1, but this time the vaccinations were tested on early-stage breast cancer patients [229]. A group of 31 patients with stage II breast cancer were injected with the vaccine formulation. This was the first evidence of a successful immunotherapy outcome of any MUC1 vaccine, as no recurrence of disease occurred over 5-year-period after the immunizations. The control group, which received only placebo, had a 27% recurrence rate. Of the 10 patients, whose samples were available for T cell proliferation assays, the T cells isolated from four patients (40%) were shown to be specific for MUC1 VNTR. Conversely, anti-VNTR serum antibodies were induced in 69% of the patients. Important findings of this study emphasize the importance of conducting immunotherapy trials on cancer patients before their immune system is compromised with the tumor burden of an advanced stage.

Other approaches to use MUC1 on immune therapeutics against breast cancer include a live recombinant vaccinia virus that expressed MUC1 and IL-2 genes [230]. This small study included nine patients with advanced breast cancer and, as opposed to the studies discussed previously, none of the patients produced high antibody titers to MUC1 after vaccination and only two had cellular responses as indicated by the production of MUC1-specific CTLs.

In addition, vaccinations with MUC1 peptide-pulsed dendritic cells (DCs) have been carried out in different trials. Metastatic breast and ovarian cancer patients that were vaccinated with autologous MUC1 peptide-pulsed DC produced CD8+ cytotoxic T cells that were detected in the peripheral blood after three vaccinations over 28 days [231].

4.2. Colon cancer

Clinical studies done on patients with resected colon carcinoma investigated vaccines containing the Thomsen-Friedenreich antigen (T) and sialyl–Tn antigen (sTn), which are overexpressed in the mucins of colorectal carcinomas [232,233]. In the first approach, 20 patients were subdivided in three groups that were immunized with partially desialylated ovine submaxillary gland mucin (modified OSM) alone, OSM administered with DETOX, or modified OSM injected with Bacillus Calmette-Guérin (BCG), respectively [232]. Although the modified OSM alone did not induced any immune response in the patients, nine out of 15 patients immunized with modified OSM and the adjuvants (DETOX or BCG) produced increased IgG titers of OSM specific antibodies, primarily against sTn. In another clinical trial, T and sTn antigens were evaluated against colorectal cancer, in an approach involving the conjugation of these antigens to the immunogenic carrier protein KLH and injection with several adjuvants in order to improve the immune response [233]. The patients injected with DETOX and QS-21 generated both IgM and IgG antibodies against the synthetic peptides. However, when analyzing the induced antibodies against natural antigens, the responses were weak to moderate for IgM and undetectable for IgG. This study demonstrates the importance of designing vaccines that will induce the immune system to recognize antigens as they are presented on a tumor environment.

Viral vaccines that expresses carcinoembryonic antigen (CEA) and MUC1 had been developed by Therion Biologics Corporation [234,235]. The PANVAC-VF consists of vaccinia or fowlpox viral vectors expressing CEA and MUC1 along with the three T cell costimulatory molecules B7.1, ICAM-1, and LAF-3 (TRICOM™). In this first National Cancer Institute (NCI)-sponsored study, 25 patients with advanced metastatic carcinoma from different tissue origins, mainly from colon, were injected with PANVAC-VF. Over half of the analyzed patients had CD8 and CD4 immune responses specific to MUC1 and/or CEA. In addition to these promising responses, a follow up of an ovarian cancer patient confirmed a duration of the clinical response for 18 months, whereas a breast cancer patient had a measurable decrease liver metastasis.

4.3. Lung cancer

As recently reviewed, cancer associated mucins are potent biomolecules to develop novel therapies against lung cancer [236,237]. One of the antigen-specific vaccination strategies investigated was the MUC1 liposomal vaccine preparation BLP25 (L-BLP25). The vaccine consists of BLP25 lipopeptide, which targets the exposed core of the MUC1 peptide, the adjuvant monophosphoryl lipid A, and three lipids including cholesterol, dimyristoyl phosphatidylglycerol, and dipalmitoyl phosphatidylcholine. In a phase I clinical trial, five of the 12 patients with advanced non-small cell lung cancer (NSCLC) that completed the vaccination regimen had cellular responses specific to MUC1 [238]. Although no antitumor responses were obtained, the higher dose injected extended the median survival time significantly. Likewise, in a phase II clinical trial with 65 NSCLC patients, 54% of the patients that received the MUC1 liposomal vaccination increased their survival for almost a year when compared with the control group [239]. In a 53 months follow-up study conducted by the same research group, the overall survival of patients receiving the L-BLP25 vaccine was further increased to 30.6 months versus the 13.3 months of non-immunized patients [240].

4.4. Ovarian cancer

High MUC1-antibody levels in ovarian cancer patients are associated with greater survival, but it does not represent a significant prognostic indicator [241]. In agreement with the reports on humoral responses to MUC1 discussed for breast cancer, IgM antibodies were detected in ovarian cancer patients with the 105-residue MUC1 peptide. In these studies, the levels of MUC1 antibodies were higher in healthy women, making unfeasible the correlation of this immune response with ovarian cancer. One of the possible explanations to these findings relies on the possible variety of immune responses against MUC1, as glycosylation patterns among different types of adenocarcinomas are not homogenous. A CTL cell line, which was derived from ovarian tumor-infiltrating or tumor-associated lymphocytes, that recognizes MUC1 positive tumors in a non-MHC restricted manner [222]. In the same study, MUC1 transfected cells were also lysed, and in this latter case it was MHC restricted. The main outcome of this study was that the CTL cell line recognizes epitopes on the core peptide of MUC1 and thus, suggested that mucin therapy can be feasible for the treatment of ovarian cancer.

A recent review published by our group, summarizes the mucin immunotherapeutic strategies against ovarian cancer [190]. One of the recent studies discussed here included a clinical trial where 11 patients with stage III-IV ovarian, fallopian tube, and peritoneal cancers were immunized with heptavalent antigens individually conjugated to KLH and administered with the adjuvant QS-21 [242]. All the mucin antigens included in the vaccine (Tn, STn, TF, Tn-MUC1) induced the higher antibody titers when compared with the nonmucin antigens and, specifically, Tn-MUC1 was able to induce both IgM and IgG responses. Refer to the review for more examples of therapies involving the mucins MUC1, MUC4, and MUC16 in ovarian cancer.

4.5. Pancreatic cancer

In a recent review describing novel therapies against pancreatic cancer, MUC1 and MUC4 are mentioned as potential targets for treatment of this malignancy [243]. One of the earlier immunogenic studies done in pancreatic cancer patients generated a cytotoxic cell line derived from lymph nodes of a patient with pancreatic adenocarcinoma, which induced MHC-unrestricted recognition of MUC1 [216]. The CTLs lysed pancreatic, breast, colon, ovarian, and other cancer cell lines that expressed mucin peptide epitopes, whereas the tumor cells lacking mucin peptide were not killed.

In a phase I clinical trial done over a decade ago, 24 pancreatic cancer patients, nine breast cancer patients, and 30 colorectal cancer patients were immunized with a MUC1 105-mer peptide that contain five immunologic epitopes [220]. The main goal of this trial was to determine the toxicities of the vaccine, using Bacillus-Calmette-Guérin (BCG) as an adjuvant, and, secondarily, to determine if the vaccine induced a hypersensitive (DHT) response specific to MUC1. Altogether, the tolerability of the vaccine was proved and 67% of the biopsies analyzed indicated a strong T cell infiltration at the injection site only when the peptide with the immunogenic epitopes was present in the formulation. Enhanced responses to MUC1 were reported in seven out of 22 patients.

In a more recent phase I study, 10 patients with advanced pancreatic cancer were immunized with the recombinant vaccinia and fowl pox viral vectors expressing CEA and MUC1, in combination with the three costimulatory molecules B7.1, ICAM-1, and LFA-3 (TRICOM™) [234]. In this study, the granulocyte-macrophage colony-stimulating factor (GM-CSF) was used as an adjuvant, and the whole vaccine formulation is known as PANVAC-VF. The prime immunizations consisted of vaccinia virus and subsequent boost injections consisted of fowl-pox virus in order to increase T cell immunity. Antigen specific T cell responses were detectable in five out of eight patients within 2 months of the immunizations. The encouraging results of the study included a significant increase in the median survival of the patients with T cell immune responses (15.1 months) when compared with the ones that did not have the T cell activation (3.9 months).

A potential immunotherapy strategy against pancreatic cancer was developed last year, where a dendritic cell-based vaccine was designed to stimulate both T cell arms of the immune system: CD4+ T helper cells and cytotoxic CD8+ T cells [244]. Dendritic cells were transduced with the universal DR-restricted T helper epitope (PADRE) combined with human leukocyte antigen HLA-A1 and HLA-A2 restricted epitopes from MUC4 using adenovirus vector. The in vitro studies showed that immunogenicity of the formulation was increased and cytotoxicity was HLA-A2 restricted and MUC4 specific. The advantage of PADRE resides on its ability to increase immunogenic responses more than three-fold when compared with natural T cell epitopes.

4.6. Prostate cancer

As recently reviewed, MUC1 shows promising results for the therapy of prostate cancer [245]. Some of these approaches for targeting MUC1 are discussed below. A vaccinia virus expressing MUC-1 and IL-2 genes was evaluated in a phase I clinical trial of previously treated patients with advanced prostate cancer, whose tumors were shown to be positive for MUC1 [246]. The progression of the disease was measured by the levels of prostate-specific antigen (PSA). Of the 16 patients enrolled in the study, only one had an objective tumor response and two showed long-term stabilization of PSA. Unfortunately, at the end of the clinical study, 14 patients had progressive disease and mild adverse effects were reported. These secondary effects are possibly caused by the IL-2 rather than the MUC-1 expression, and to implement the vaccine for future studies, more modifications need to be done in order to minimize these undesirable effects while increasing its immunogenicity.

As discussed for patients with lung cancer, the liposomal vaccine BLP25 also shows promise for prostate cancer patients as demonstrated in a small clinical trial of 16 patients [247]. Even though the study was of small size, the liposomal vaccine prolonged the protein specific antigen (PSA) doubling time in half of the patients, which may delay the clinical relapse of the disease.

4.7. Renal cancer

In a study including patients with advanced cancer immunized with oxidized mannan conjugated to MUC1 VNTR, demonstrated that the vaccine did not induce antibody responses but only cell responses [248]. This trial included patients with renal, colon, breast, and ovarian among other types of cancer. IFNγ-secreting T cells specific to the VNTR MUC1 region were identified in the 10 patients included in the study, whereas nine out of them had DTH responses. Even though the anti-tumor response was not proven, the immune responses were still detectable after a year of the last immunization.

In another promising phase I trial with metastatic renal cancer patients, 30% of the MUC1- immunized patients had a noticeable regression of metastasis [249]. The vaccine consisted of autologous DCs pulsed with MUC1 peptides and the T helper epitope PAN-DR-binding peptide (PADRE) was included as an adjuvant therapy. Cytotoxic T cell responses were detected roughly in half of the patients after three to four vaccine injections. After five immunizations, the patients were treated with low doses of IL-2 as part of the combination therapy. It is important to mention that the immunologic responses discussed previously were detected before the treatment with IL-2. With these promising results, the same group published a short review suggesting the potential of MUC1-pulsed DCs to induce immunological and clinical responses in patients with advanced renal cell carcinoma [249].

5. Mucins as conjugated therapeutics

Because they are differentially expressed in malignant tissues, mucins have been investigated for targeted therapies. Several approaches include conjugating mucin antibodies to radioactive elements and/or cytotoxic drugs. Once the distinct epitopes of mucins are targeted by the conjugated antibodies, the malignant cells are selectively destroyed by radiation and/or the cytotoxic drug.

5.1. Breast cancer

Work that was published this year describes a novel approach against epithelial cancer cells [250]. The potential therapy consisted of using phototoxic single stranded DNA aptamers that display a peptide corresponding to five MUC1 tandem repeats modified with up to 15 GalNAc sugars (Tn antigen). The specific entry of the phototoxic aptamers to MUC1 positive cancer cells (breast, colon, lung, ovarian, and pancreatic) occurred by receptor-mediated endocytosis. The toxicity was increased over 500-fold after light activation when compared with the drug alone. Even if this work was demonstrated on in vitro experiments, it shows the potential of using mucins to deliver cytotoxic drugs, such as photodynamic therapy drugs into cancer cells to eradicate them.

Another technique that is currently being developed is a MUC1 pre-targeting molecule for imaging and pre-targeting radioimmunotherapy of metastatic breast and prostate cancers [251]. The three major components of the pre-targeting molecule are a tumor antigen binding module or single chain antibody fragments (scFv), a radioactive hapten capturing molecule, and a bifunctional polyethylene glycol (PEG) for the covalent attachment of both. Preclinical studies are being performed and different valency of the antibody fragments is being compared on in vivo experiments to determine the best formulation.

5.2. Ovarian cancer

In a randomized phase III study, patients with ovarian cancer were treated with intraperitoneal radioimmunotherapy using 90Y-HMFG1 monoclonal antibody targeting MUC1 [252]. The patients that entered the study with complete remission after chemotherapy had a median survival of 78% over 10 years of follow up studies. In a similar study published more recently, the same radiolabeled murine antibody was analyzed in a phase III clinical trial to determine the immunogenicity of the drug in ovarian cancer patients and the outcome of the disease [224]. It was found that MUC1 IgG antibodies were induced in the patients, but no difference in disease outcome was observed in the patients that did not receive the injections. However, when patients receiving the drug were compared, the ones that had greater levels of MUC1 antibodies had an improved disease outcome than those that did not respond to the vaccine.

5.3. Prostate cancer

A combination therapy of 111In/90Y-monoclonal antibody, m170, which targets expressed MUC1, and paclitaxel given to patients with metastatic prostate cancer increased the therapeutic index of targeted radiation therapy [212]. The metastatic cancer was successfully targeted in 25 out of 26 patients, indicating the specificity of MUC1 recognition in prostate tumor cells. This study confirmed an increased efficacy of targeted radiation when compared to previous studies done with radioimmunotherapy alone.

6. Other approaches for the use of mucins as valuable therapeutic tools

Clearly, numerous studies discussed in this review have shown that mucins can be successfully recognized when injected as a vaccine and the focus has been mainly to target cytotoxic T cells. However, some studies emphasize the importance of developing vaccines that prime not only the cytotoxic T cell responses but also the activation of mucin-specific helper T cells, in concordance with the hypothesis that a cancer vaccine should prime the activation of both arms of the immune system [253]. One of the studies that emphasizes the activation of humoral responses or helper T cell responses has just been published [254]. The strategy evaluated in murine models indicates that the induction of antibodies specific to sialylated Thomsen-noveau (STn) antigens, which can be carried on various mucins, created significant delay in tumor growth. Similarly, in other study performed in a MUC1-tolerant colon cancer murine model, the vaccine included MHC class I-restricted MUC1 peptides as well as MHC class II-restricted pan helper peptide [255]. In addition, the vaccine formulation included unmethylated CpG oligodeoxynucleotide, and GM-CSF as adjuvants. This promising study was successful in breaking MUC1 self-tolerance in MUC1.Tg mice, both prophylactically and therapeutically.

Other in vitro approaches involved the development of self-inactivating lentiviral vectors encoding the human MUC1-specific MHC-unrestricted single chain T cell receptor and a fusion suicide gene to eradicate transduced cells in a safe manner [256]. This represents a novel and safe approach to target MUC1 in malignant tissues to activate innate and adaptive immune responses.

In addition, studies involving other mucins have shown promise for further clinical analysis. One such examples is an anti-idiotype MUC16 antibody fused with IL-6 that induced specific humoral responses against MUC16 (CA125) in ovarian cancer [257].

7. Future perspectives

More than 200 clinical studies have been done over the last three decades to evaluate mucins as prognostic indicators or therapeutic constituents. The current need to find alternative treatments against cancer and the promise of mucins is denoted by the current clinical trials involving these glycoproteins in observational as well as in interventional therapeutics studies against cancer. Nevertheless, it is important to keep in mind that outcomes obtained from clinical trials done in patients with advanced cancer might underestimate the therapeutic value of new vaccine formulations. Altogether, there is no doubt that these macromolecules represent a potential tool for the diagnosis and therapy of a wide range of cancers and their incorporation into clinical medicine represents an advance in the continuous search for the cure of cancer. Advances in our knowledge on splice variants, domain structrures, development of new MUC specific antibodies, and characterization of glycosylations on these proteins will form the basis for new clinical trials.