ABSTRACT
Mucins are high molecular weight glycoproteins forming the mucus that coats the surface of human epithelial cells lining mammary glands, salivary glands, digestive tract, respiratory tract, and urogenital tract (1). The major function of mucins is to protect epithelial cells from dehydration, infection, and other physical or chemical injuries.Expression of mucins often changes during the malignant transformation of epithelial cells (2,3). Mucin synthesis may increase markedly, resulting in loss of cellular polarity. Large amounts of mucus are shed or secreted by tumor cells. Furthermore, diverse patterns of glycosylation changes on mucoprotein peptide backbone usually occur, resulting in the expression of novel carbohydrate epitopes or the exposure of peptide backbone (4,5). Alteration of mucin glycoprotein may be involved in several stages of metastasis, including cell growth, motility during invasion, interactions between tumor cells, platelets, and the immune system, and adherence of tumor cells to endothelia and extracellular matrix (6). Aberrant expression of carbohydrate epitopes or mucin core proteins has been shown to negatively influence the outcome of several neoplasms, such as colorectal cancer (7-9). 265
The dysregulation of mucin expression in cancer includes aberrant glyco-sylation, underglycosylation, and overexpression of mucin peptides (10-12). In vitro studies have demonstrated that expression of mucin in cancer cells can decrease tumor cell aggregation, promote tumor cell invasion, and block lymphocyte targeting, thereby facilitating metastasis by escaping systems of immunosur-veillance. Highly metastatic colon carcinoma cells express significantly more sialylated glycoproteins than poorly metastatic cells. The expression of sialylated antigens may “ mask” recognition in tumor metastasis by T and natural killer cells in the immune system. Increased expression of sialyl Lewis x on cell surface glycoproteins can serve as better ligands for selectins present on endothelial cells and paltelets (13-16). Moreover, high mucin-producing cancer cells may secrete more proteolytic enzymes, such as collagenase, than low mucin-producing cells. Collagenase activity is further stimulated by purified mucin, thus promoting tumor invasion (17). MUCIN GENES AND AIRWAY MUCINS
To date, at least 10 mucin genes have been completely or partially cloned, and they can be grouped into two types: membrane-bound and secreted. MUC1 and MUC4 are membrane-bound mucins (18,19), while MUC2, MUC5AC, MUC5B, and MUC7 are secreted mucins (20-23). The others, MUC3, MUC6, MUC8, and MUC9, remain unclassified (24-27).MUC1, the gene encoding episialin, a membrane O-glycoprotein, has been located on chromosome lq21-24. MUC2 and MUC3, genes coding for secreted intestinal mucins, were mapped to chromosomes llq l5 and 7q22, respectively. Tracheobronchial mucin cDNAs were used to identify three mucin genes, MUC4, MUC5AC, and MUC5B, which have been mapped to chromosome 3q29 (MUC4) and chromosome llp l5 (MUC5AC and MUC5B). MUC6 gene was cloned and identified in stomach and gall bladder and was mapped to chromosome llp l5 . MUC7, expressed in salivary glands, has been localized to chromosome 4. MUC8, expressed in both goblet cells and submucosal glands of human trachea, was mapped to 12q24.Of the 10 human mucin genes, seven (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7, MUC8) are expressed in human airway epithelium, as demonstrated by Northern blot analysis and in situ hybridization (Table 1). However, the exact role of each of the seven mucin gene remains unclear (28-32). MUC1 is expressed in the primary culture of human nasal and tracheobronchial epithelial cells but not in squamous metaplastic epithelia, implying its close relationship with mucus differentiation of epithelial cells (33). MUC2 is known to exist in several cell types of the respiratory tract. However, it has not been observed in cultured airway epithelial cells and is not considered to be a major mucin gene
in human respiratory mucosa (34). As previously stated, MUC4, MUC5AC, and MUC5B had been cloned from tracheobronchial mucin cDNA library. MUC4 is located in both ciliary and goblet cells and is considered to be the very first mucin gene expressed in the developmental process of human respiratory tract. In contrast, MUC5AC and MUC5B are expressed later in the developmental phase of the airway. Biochemical analysis has identified MUC5AC and MUC5B as major contributors to the mucin produced by human airway epithelium (35-37). Among three major gel-forming mucins in human sputum obtained from patients who died of asthmaticus, two are differently charged glycoforms of MUC5B, while the other is MUC5AC. And, in contrast to MUC5AC, which originates mainly in goblet cells (Fig. la), MUC5B is the major mucin product of mucous cells of submucosal glands. Although both MUC7 and MUC8 are produced by submucosal glands, they are unlikely to be the major secreted airway mucin (32). AIRWAY MUCIN AND LUNG CANCER: EXPRESSION PATTERNS
So far, research investigating the expression patterns of mucin genes and their products in lung cancer has been limited. The expression patterns of mucin genes in cancer cell lines are different from those in cancer tissues. Berger et al. studied the expression patterns of five mucin genes (MUC1, MUC2, MUC4, MUC5AC, and MUC5B) in six lung cancer cell lines by Northern and Western blot analyses (38). They demonstrated variable expression patterns of mucin genes in different cell line. All cell lines expressed MUC1 mucin, but none expressed MUC2. Calu-3 and A549 cell lines expressed high to moderate amounts of MUC5AC mucin, whereas NCI-H292 cells did not express as much. On the other hand, H292 cells expressed the highest amount of MUC4 in all cell lines.By studying lung cancer tissues, Nguyen et al. found that lung adenocarcinomas, especially well-differentiated cancers, exhibited increased MUC1, MUC3, and MUC4 mRNA levels. Yet, squamous-cell, adenosquamous, and large-cell carcinomas were characterized only by increased levels of MUC4 mucin (39).Another study evaluating the expression patterns of mucins in lung cancer tissue samples was performed by Seregni et al. (40). Their results showed that the intensities of MUC1 and MUC4 expressions were always higher in cancer tissues than in normal cell lines. Similar to the study by Nguyen et al., the highest reactivity for MUC1 and MUC4 expression was observed mainly in the adenocarcinoma histotype, which is mucin secreting.In one of our previous studies, we examined the expression patterns of mucin genes in 7 lung adenocarcinoma cell lines and 12 lung adenocarcinoma tissues. We performed Northern blot analysis with specific antisense oligonucleo-
tide probes that recognize mucin-specific tandem repeats of four mucin genes (MUC1, MUC2, MUC3, MUC4) and carried out RT-PCR to amplify the 3' and 5' nonrepetitive coding region of MUC1 and the 5' nonrepetitive coding region of MUC2 (41). Of the seven cell lines, CL1 and PC 13 are poorly differentiated with low mucin glycoprotein production (42), while the rest are well differentiated. All cell lines expressed MUC1, MUC2, MUC3, and MUC4 mRNAs, but in variable quantities. The poorly differentiated cell lines (CL1 and PC 13) had a relatively low level of expression of MUC1-4. RT-PCR also revealed the presence of MUC1 and MUC2 mRNA in all the cell lines.The expression pattern of mucin genes is consistent with that of mucin glycoproteins as studied using biochemical and immunological methods. Northern blotting and RT-PCR analyses in 12 lung adenocarcinoma tissues with various grades of differentiation (6 poorly differentiated adenocarcinoma and 6 moderately to well-differentiated adenocarcinomas) showed heterogeneous expressions of the four mucin genes in tissues without clear correlation with the differentiation grade.We next studied mucin expression patterns in paired lung cancer and nontu-morous lung portions (43). Sixty tissues pairs with varying types and stages were included. Slot blot analyses with specific antisense oligonucleotide probes derived from tandem repeat sequence of MUC1, MUC2, MUC3, MUC4, MUC5B, and MUC5AC were utilized to compare the amount of mucin gene mRNA in tumor samples with that of the nontumor counterparts. A ratio higher than 1.5 for each specific mucin mRNA amount was considered to indicate mucin gene overexpression in tumors. The study showed that mucin gene overexpressions frequently occurred in lung cancer (25 out of 60, 41.7%); MUC1 overexpression was found in 7 tumors (11.7%), MUC2 in 5 (8.3%), MUC3 in 12 (20%), MUC4 in 9 (15%), MUC5B in 10 (16.7%), and MUC5AC in 13 (21.7%). Immunohisto-chemical stain demonstrated the expression of mucin product in lung cancer tissue (Fig. lb) However, there was no preferential expression of any particular or combination of mucin genes in lung tumors. Overexpression of mucin genes and mucin proteins appeared to have no correlation with tumor stage, nodal stage, histology, or pathological differentiation grade. AIRWAY MUCIN AND LUNG CANCER: AS A PROGNOSTIC FACTOR
Lung cancer is one of the most commonly diagnosed malignancies throughout the world. The prognosis of lung cancer is generally poor. Only 20-30% of the patients are operable at diagnosis, and only half of these can be successfully resected. The 5-year survival rate of lung cancer is less than 15%. About 80% of the patients die of tumor recurrence following surgical resection (44,45).
