State of the Art: Concise Review
Mucins in Lung Cancer Diagnostic, Prognostic, and Therapeutic Implications Imayavaramban Lakshmanan, PhD,* Moorthy P. Ponnusamy, PhD,* Muzafar A. Macha, PhD,* Dhanya Haridas, PhD,* Prabin Dhangada Majhi, PhD,* Sukhwinder Kaur, PhD,* Maneesh Jain, PhD,* Surinder K. Batra, PhD,*†‡ and Apar Kishor Ganti, MD, MS§║
Abstract: Aberrant expression of mucins is associated with cancer development and metastasis. An overexpression of few mucins contributes to oncogenesis by enhancing cancer cell growth and providing constitutive survival signals. This review focuses on the importance of mucins both in the normal bronchial epithelial cells and the malignant tumors of the lung and their contribution in the diagnosis and prognosis of lung cancer patients. During lung cancer progression, mucins either alone or through their interaction with many receptor tyrosine kinases mediate cell signals for growth and survival of cancer cells. Also, stage-specific expression of certain mucins, like MUC1, is associated with poor prognosis from lung cancer. Thus, mucins are emerging as attractive targets for developing novel therapeutic approaches for lung cancer. Several strategies targeting mucin expression and function are currently being investigated to control lung cancer progression. Key Words: Lung cancer, Mucins, MUC1, MUC4, MUC5AC, MUC7, Mucin vaccines, L-BLP25, TG4010, Aptamer. (J Thorac Oncol. 2015;10: 19–27)
M
ucins are high molecular weight, heavily glycosylated proteins that are expressed in the epithelial cells of various organs. The presence of a variable number of tandem repeats in the extracellular domain distinguishes mucins from other glycoproteins. The number of tandem repeats is unique to each mucin and contains several proline, threonine, and serine (PTS) residues, but is usually devoid of cysteine residues. This mucin domain/PTS region provides the acceptors for relatively short O-linked glycans (2–20 monosaccharides per chain).1 The PTS region forms the densely packed filamentous carbohydrate-rich moiety that determines most of the biophysical properties of
*Department of Biochemistry and Molecular Biology, †Department of Pathology and Microbiology, ‡Eppley Institute for Research in Cancer and Allied Diseases, §Department of Internal Medicine, VA NebraskaWestern Iowa Health Care System, Omaha, Nebraska, and ║Division of Oncology–Hematology, Department of Internal Medicine, University of Nebraska Medical Center, Omaha,Nebraska. Disclosure: The authors declare no conflict of interest. Address for Correspondence: Apar Kishor Ganti, MD, MS, FACP, Division of Oncology–Hematology, Department of Internal Medicine, University of Nebraska Medical Center, 987680 Nebraska Medical Center, Omaha, NE 68198. E-mail: [email protected] DOI: 10.1097/JTO.0000000000000404 Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/15/1001-0019
mucins. Mucins are classified into two major groups: membranebound mucins and the secretory mucins (Fig. 1). There are 11 membrane bound mucins, namely, MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC20 and MUC21; and seven secreted mucins, MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC8, and MUC19.2 Mucins are expressed during embryonic stages of lung development and are involved in the normal development of the lung. Several reports suggest that mucins are overexpressed in non–small-cell lung cancer (NSCLC) and can be used as suitable biomarkers for identifying and monitoring the progression of lung cancer.3–6 Under normal conditions, mucins serve as a protective barrier for epithelial cells of various organs including lung.7 Deregulation of membrane-bound and secretory mucins is associated with cancer progression and metastasis by altering various signaling pathways.8–10 Of the various mucins, membrane-bound mucin MUC1 and secretory mucin MUC5AC seem to be predominantly found in NSCLC and are correlated with disease progression. Lung cancer is the most common cause of cancerrelated deaths in the United States.11 Mucins have not been extensively studied for their role in the pathogenesis of lung cancer. This may be due to the complexity of mucin biology and the existence of multiple mucins with differing functions within the cell. However, as our knowledge of mucins in other diseases is increasing, the role of various mucins in lung cancer too will be understood better. This review addresses the current knowledge about the expressional status and role of mucins in lung cancer cell, their significance as prognostic and diagnostic markers and their potential as therapeutic targets.
EXPRESSION AND FUNCTION OF MUCINS IN NORMAL LUNG EPITHELIUM Mucins secreted by the goblet cells in the respiratory epithelium form the mucosal coat of the apical epithelium and thereby protect the airways and alveoli from insults such as dehydration, pathogen invasions, and chemical agents.12 In a normal airway, expression of MUC1 is weak, and a diffuse signal is observed in the tracheobronchial and collecting duct epithelium. MUC1 expression is not observed in the submucosal glands and bronchioles.13 Weak MUC2 expression is observed in the entire respiratory epithelium; however, the basal pole of some goblet cells expresses MUC2 moderately.13 MUC2 is undetectable in
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
19
Lakshmanan et al.
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
FIGURE 1. Structure of membrane-bound and secretory type mucins and their domain. Schematic representation of the most differentially expressed membrane-bound (MUC1 and MUC4) and secretory mucins (MUC5AC) in lung cancer and their associated O-glycans: lung cancer is characterized by differential expression of MUC1 and de novo expression of MUC4 and MUC5AC. Domain structure of MUC1: The epitome of membrane-bound mucins, MUC1, is characterized by the presence of an N-terminal sequence, 20 tandem repeat domains (20–120 amino acid each), single self-cleavable sea-urchin sperm protein, enterokinase, and agrin (SEA) domain (two subunits of MUC1 are generated by auto-proteolysis at the SEA domain that, in turn, forms a stable noncovalent heterodimer), single spanning TM domain and 72 amino acid (aa) cytoplasmic tail (CT) that contains binding sites for various effector molecules including ERα, p53, GSK-3β, PKCδ, cSRC, and β-catenin. The MUC1 cytoplasmic tail is target of various oncogenic signaling pathways. Domain structure of MUC4: De novo-expressed transmembrane mucin MUC4 is putatively cleaved at Gly-Asp-Pro-His (GDPH) proteolytic site generating an extracellular subunit MUC4α and the membrane-tethered subunit MUC4β. MUC4α is characterized by a unique N-terminal sequence, three imperfect repeats, heavily O-glycosylated tandem repeat domain of 16 amino-acids, a cysteine-rich domain (CRD), a nidogen (NIDO) domain having similarity to the nidogen-epidermal growth factor (EGF) domain of ancestral nidogen proteins, eight invariant cysteine residues containing the adhesion-associated domain in MUC4 and other proteins (AMOP) domain. MUC4β is a growth factor-like subunit characterized by the presence of three EGF like domains, a vWD-domain having similarity to dimerization domains of the von Willebrand D (VWD) factor, a membrane-spanning transmembrane domain and a short cytoplasmic tail of 32 amino acids. Domain structure of MUC5AC: MUC5AC, a differentially expressed gel-forming mucin, has N- and C-terminal domains with high similarity to cystein-rich domain that is involved in disulfide-linked multimer formation. It includes disulfide-rich D-domains (D1, D2, D’, and D3) at the N-terminus and D4, B, C, and CK (vWF-like domain) at the C-terminus. The central region is characterized by CRDs (n = 17, aa = 110 having 10 cysteine residues). The first nine CRD are interspersed by threonine/serine/proline-rich domains (TSP) with no repetitive sequences, whereas Cys5 to Cys9 domains are interspersed with highly O-glycosylated TR domains (N = 4). Through its CRDs and CK domain, it forms disulfide-linked dimers and oligomers. Furthermore, its C-terminus has a GDPH autocatalytic proteolytic site.
the bronchioles and alveolar epithelial cells.14 In contrast, most of the respiratory epithelial cells including the basal, ciliated, and goblet cells as well as the collecting ducts express MUC4.15
20
Strong expression of MUC4 is found in the collecting ducts of the sub-mucosal glands and alveolar epithelial cells.14 Interestingly, MUC4 is one of the first mucins expressed in the early stages
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
of embryonic development in the lungs (as early as 6.5 week after gestation),13 and it is speculated to be involved in the differentiation of all respiratory epithelial cells.13 After 12 weeks of gestation, MUC4 is observed in the small bronchi and bronchioles of lungs (Table 1).13,15 Moderate expression of MUC5AC is initially observed at the 13th week of gestation in the epithelial buds of the segmental bronchi. MUC5AC is strongly expressed in the trachea and main bronchi, but no expression is seen in the bronchioles and smaller alveolar epithelial cells.14 MUC5AC is also expressed in the goblet cells of the surface epithelium and in the glandular ducts.14 MUC5B starts to appear after the 13th week of gestation and a weak expression is observed in the bronchial epithelium, mucous cells of the sub-mucosal glands, and glandular ducts.13,15 MUC5B is not expressed in the bronchioles and alveolar epithelial cells, but is strongly expressed in the collecting ducts and mucous cells of sub-mucosal glands.16 MUC7 transcript is detected only in the serous cells of some sub-mucosal glands.15 Other mucins such as MUC3 and MUC6 are not expressed in the normal respiratory mucosa.15 In summary, mucins exhibit distinct spatiotemporal expression pattern in different regions of the respiratory epithelium and during lung development (Table 1).
MUCINS IN LUNG CANCER MUC1 In normal epithelial cells, MUC1 expression is restricted to the luminal side of the cells.17 However, during progression of lung cancer, there is a loss of cell polarity with resulting MUC1 expression over the entire cell surface.18 MUC1 expression has been classified into three types, high-grade polarized expression (HP), low-grade polarized expression (LP), and depolarized expression (DP). HP is defined as having polarized expression of MUC1 in greater than 50% tumor cells and less than 10% cells showing depolarized expression of MUC1. LP is less than
Mucins in Lung Cancer
50% of cells with polarized expression of MUC1 and less than 10% of depolarized expression of MUC1, whereas DP is considered as depolarized MUC1 expression in greater than 10% of cancer cells.5,19 Based on this classification, HP of MUC1 was seen in well-differentiated adenocarcinoma, whereas depolarized expression of MUC1 correlated with increasing stage and lymph node metastasis.20,21 Depolarization of MUC1 expression extends from the apex and moves across the entire surface thereby preventing cell–cell and cell–matrix interaction, thereby resulting in increased invasion into adjacent tissues. Differential polarized expression of MUC1 was also observed between primary and pulmonary metastatic tumors. HP and DP of MUC1 were significantly higher in primary lung cancer tissues than in that of metastatic lesions.5 Increased depolarized expression of MUC1 also correlated with progression of atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC; adenocarcinoma with lepidic pattern in the new International Association for the Study of Lung Cancer [IASLC] classification).3 The clinicopathological significance of MUC1 was analyzed in 126 patients with primary lung cancer and 147 patients with metastatic tumors in the lung. The (18)F-FDG uptake was higher in patients with depolarized expression (DP) as compared with namely HP or LP. HP and DP pattern of MUC1 was seen in a higher proportion of patients with primary lung tumors as compared with metastatic lesions but LP of MUC1 was detected in a higher proportion of patients with metastatic lesions.5 The study suggested that depolarized expression of MUC1 was associated with poor prognosis. These findings suggest that differential distribution of MUC1 plays a significant role in aggressiveness of lung cancer and metastasis. MUC1 signaling and its role in aggressiveness of lung cancer cells MUC1 overexpression induces several downstream signaling pathways that are closely associated with poor clinical
TABLE 1. Mucins Expression in Normal Bronchial Cells and Carcinomas Mucins
Expression in Normal Respiratory Tract
MUC1
Trachea bronchial epithelium, collecting ducts, submucosal gland and bronchioles13
MUC4
Goblet cells, collecting duct, and alveolar epithelial cells13,15
MUC2 MUC5AC MUC5B
Respiratory epithelium, goblet cells13 Surface epithelium of goblet cells, trachea, and main bronchi13,14 Bronchioles and alveolar epithelial cells, collecting duct and submucosal gland13,15 Serous cells of submucosal gland.13,15
MUC7
Expression in Preinvasive and Invasive Lesions Preinvasive:54 All preinvasive lesions: 77% (34 of 44) Squamous dysplasia: 83.3% (20 of 24) Bronchiolar columnar cell dysplasia: 83.3% (5 of 6) Basal cell dysplasia: 66.6% (4 of 6) Bronchial epithelial dysplasia with transitional differentiation: 66.6% (2 of 3) Columnar cell dysplasia: 66.6% (4 of 6) Invasive:54 Squamous component: 96.7% (30 of 31) Adenocarcinoma components: 87% (27 of 31) Adenocarcinomas: 81% (151 of 187)6 Squamous cell carcinomas: 78% (69 of 88) Adenosquamous carcinomas: 75% (6 of 8) Large cell carcinomas: 55% (33 of 60) No data Stage I NSCLC: 16 of 61 (26.2%)74 No data No data
BCCD, bronchiolar columnar cell dysplasia; BCD, basal cell dysplasia; BEDT, bronchial epithelial dysplasia with transitional differentiation; CCD, columnar cell dysplasia; NSCLC, non–small-cell lung cancer.
Copyright © 2014 by the International Association for the Study of Lung Cancer
21
Lakshmanan et al.
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
outcomes.22 Overexpression of MUC1 favors angiogenesis by upregulating vascular endothelial growth factor (VEGF) expression through activation of the Akt and ERK signaling pathways. Giatromanolaki et al.23 demonstrated that highly vascularized NSCLC tumors had high MUC1 expression, and this appeared to correlate with VEGF expression. MUC1 is also involved in the inactivation of integrin-mediated cell-to-matrix adhesion and E-cadherin mediated cell-to-cell adhesion.24 MUC1 knockdown led to sensitization of NSCLC cell lines to cisplatin treatment and elevated apoptosis due to reduced Bcl-XL activity.25 MUC1 has been shown to interact with EGFR and β-catenin through its cytoplasmic domain and promote cancer cell proliferation.26 MUC1 cytoplasmic domain contains 72 amino acids; a 15 amino acid sequence is responsible for interaction with EGFR (YEKV) and β catenin (SAGNGGSSLS).27 Bitler et al.28 synthesized a MUC1 inhibitory peptide to block these interactions along protein
transduction domain PTD4 for enhancing the cellular uptake. MUC1 inhibition in lung cancer cells led to decreased proliferation by affecting transcription of the estradiol-activated reporter genes, endogenous cyclin D1, and nuclear respiratory factor-1.29 MUC1 C-terminal (MUC1-C) subunit forms a complex with EGFR by interacting with Galectin-3.30 In addition, the MUC1-C acts as a substrate for c-Src phosphorylation in lung cancer cells.27 Recently, MUC1-C was reported to be aberrantly expressed in NSCLC and through its association with p85 subunit of PI3K was found to enhance the Akt signaling pathway.31 MUC1 undergoes oligomerization through its CysGln-Cys (CQC) motif present on its cytoplasmic domain and promotes cancer cell proliferation. The MUC1 inhibitor (GO-201) inhibits MUC1 oligomerization by binding to the cytoplasmic domain and reduces MUC1-mediated cell proliferation (Fig. 2).32 In addition, other MUC1-specific
FIGURE 2. Differential signaling of mucins in lung cancer pathogenesis. Overexpression of MUC1 activates various signaling pathways for lung cancer cell proliferation through PI3K and Akt activity, MUC1 also participates in angiogenesis through regulation VEGF expression. MUC1 and EGFR signaling also promote lung cancer cell proliferation through Src signaling pathways. Similarly, secretory mucin MUC5AC promotes lung cancer cell proliferation by alterating Ras signaling pathways and cytokines. Several inhibitors are available to inhibit the lung cancer cell proliferation either by directly targeting MUC1 by L-BLP25, G0203, or by Mucins downstream target such mTor and ERK1/2 and Akt. EGF, epidermal growth factor; mTOR, mammalian target of rapamycin; ER-α and β, estrogen receptor α and β; VEGF, vascular endothelial growth factor; SRC-v-src, aviansarcoma.
22
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
inhibitors such as GO-202 and 203 were also associated with an impaired proliferation of lung adenocarcinoma cells.29 Blocking the oligomerization of MUC1-C by G0-203 inhibited NSCLC cell growth and survival by preventing the interaction between MUC1-C and PI3K-p85. This resulted in the inhibition of Akt phosphorylation and mammalian target of rapamycin activity (Fig. 2).32
Mucins in Lung Cancer
MUC1 gene can be regulated by various signaling molecules and transcription factors in the airway epithelial cells and lung cancer cells. STAT3 is highly overexpressed in NSCLC and its knockdown significantly downregulates MUC1 expression, indicating that STAT3 regulates MUC1 gene expression in lung cancer cells (Fig. 3).8 Mikami et al.25 showed that hypoxia induced MUC1 expression in lung
FIGURE 3. Regulation of mucin genes through various signaling pathways in lung cancer cells: The transcription factor STAT3 regulates MUC1 gene expression in lung cancer cells under hypoxia condition through HIF1-α.8 Further MUC1gene is also regulated by microRNA149 in lung cancer cells.34 TNF-α–converting enzyme (TACE) also regulates both MUC1 and MUC5AC in lung cancer cells. For MUC1 gene expression, neutrophil elastase may activate Protein kinase C δ for expression of dual oxidase 1 (Duox1) that leads to reactive oxygen species production for TACE activation. Then TACE induced MUC1 gene expression through TNF-α/TNF-α-Receptor/ERK1/2/SP-1 transcription factor (Pathway A). For MUC5AC gene expression, chemokine (C–C motif) ligand (CCL20) binds with GPCR complex through EGFR activation. Then TACE induce the MUC5AC gene expression through ERK1/2 signaling (Pathway B). Notch1 upregulates EGFR expression that leads to MUC5AC gene expression through ERK signaling.44 (Pathway C.) In addition, MUC5AC gene can also be regulated by microRNA-378 in lung cancer and its expression positively correlates with MUC5AC gene expression.49 MUC2 and MUC6 genes may be regulated by CDX transcription factors in lung cancer cells. Collectively EGFR and its downstream signaling molecule ERK1/2 are necessary for mucin gene expression in lung cancer cells. STAT3, signal transducer and activator of transcription 3; EGFR, epidermal growth factor receptor 1; TGF-α, transforming growth factor-α; ERK1/2, extracellular signal-regulated protein kinases 1 and 2; HIF1α, hypoxiainducible factor 1-α; CCL20-Chemokine (C–C motif) ligand 20; GPCR, G protein-coupled receptors; TACE-TGF-α, tumor necrosis factor-α–converting enzyme, CDX1 and 2, caudal type homeobox 1&2. Similarly, MUC2 and MUC6 genes can be regulated by CDX molecules as well as SP-1. Copyright © 2014 by the International Association for the Study of Lung Cancer
23
Lakshmanan et al.
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
adenocarcinoma cells by means of HIF-1α, and was partially responsible for lung cancer progression. Neutrophil elastase regulates MUC1 mucin gene expression through Sp-1 transcription factor in lung cancer cells. Specific binding of Sp-1 on the MUC1 gene promoter between -99/-90 induced gene transcription (Fig. 3).33 Similarly, the same group has elucidated the detailed signaling mechanism of Sp-1-mediated MUC1 gene expression, e.g., neutrophil elastase can activate protein kinase δ and dual oxidase 1 (Duox1) that leads to reactive oxygen species production for TNFα-converting enzyme (TACE). TACE, in turn, activates TNFα and its receptor TNFR and results in MUC1 gene expression through MEK1/2, ERK1/2, and Sp-1 (Fig. 3). This study suggests that the anti-inflammatory role of MUC1 is controlled by inflammatory mediators, such as neutrophil elastase and TNFα.33 In addition, microRNA 149 has shown to repress MUC1 expression in lung adenocarcinoma.34 Thus, MUC1 is overexpressed in lung carcinoma and its expression is tightly regulated by a number of inflammatory regulators (Fig. 3).
MUC4 MUC4 is aberrantly overexpressed in many tumors including lung carcinoma.35 Membrane-bound MUC4 is widely expressed in NSCLC, but its expression varies between different histologic subtypes. Adenocarcinoma cells express more MUC4 than squamous cell carcinoma and large cell carcinoma cells.6 MUC4 expression was detected in the pleural infiltration zone and has been shown to be required for lung adenocarcinoma development.36 Karg et al.37 reported a significant association of MUC4 expression with HER2/ERBB2 that resulted in apoptosis and differentiation rather than lung cancer cell proliferation. In contrast, other studies have demonstrated that MUC4 expression was unrelated to MUC1, HER2, and p27 expression, thereby suggesting that MUC4 can be used as an independent prognostic marker for lung adenocarcinoma.38 Recently, we demonstrated that MUC4 expression was associated with decreased proliferation, motility and invasion of lung cancer cells by attenuating the activation of Akt, Cyclin D1, and Cyclin E39 (Fig. 2). In lungs, MUC4 gene expression is controlled by IL-9 in a time- and dose-dependent manner; Jak3 appears to be involved in IL9-mediated MUC4 gene expression (Fig. 3).40 MUC4 seems to also be controlled by an epigenetic mechanism. When the DNA methylation status of 94 CpG sites from −3622 to +29 was analyzed using a MassARRAY analysis, the MUC4 expressing H292 lung cancer cell line had low methylation levels (near the transcriptional start site) and hypomethylation at the CpG sites 108 to 112 was associated with MUC4 expression.41 Similarly, when non-MUC4 expressing A427 lung cancer cells were treated with hypomethylating agents, 5-aza-2′-deoxycytidine and trichostatin A, there was an increase in the MUC4 mRNA expression suggesting that methylation status may regulate MUC4 expression. Secretory mucins MUC5AC, MUC5B, MUC2, MUC6, and MUC7 Secretory mucins protect tumor cells from exogenous cytotoxicity or genotoxicity and contribute to their
24
aggressiveness. Mucinous adenocarcinoma, a more aggressive subtype of lung adenocarcinoma, has a high expression of MUC2, MUC5AC, and MUC6. The aggressiveness of the tumor may be dependent on the mucins it secretes.42 Of all the mucins in airway epithelium, the regulation of MUC5AC is most well studied. Human MUC5AC expression is regulated by chemokine (C–C motif) ligand (CCL20)/ GPCR complex by EGFR activation by means of metalloprotease TACE.43 A recent study has elucidated two different mechanisms of EGFR-mediated MUC5AC synthesis and secretion in H292 lung cancer cells: (1) EGFR phosphorylation upon binding to TGF-α results in the production of MUC5AC and CCL20 by means of ERK activation, and (2) EGFR phosphorylation is directly activated by TACE which cleaves the proligands specific for EGFR43 (Fig. 3). In atypical adenomatous hyperplasia (AAH) and BAC with mixed subtypes (classified as “adenocarcinoma, predominantly invasive with some nonmucinous lepidic component” in the 2011 IASLC/ATS/ERS classification of adenocarcinoma), cells express high levels of secretory mucins such as MUC2, MUC5AC, and MUC6. The expression of MUC2 and MUC5AC are controlled by epidermal growth factor family and mitogen-activated protein kinase (MAPK) cascade.14 MUC2 expression was also associated with wild type p53, but not mutant p53. Treatment of H292 lung cancer cells with EGF and TNF-α resulted in increased expression of MUC2 and MUC5AC but there was no change in MUC5B and MUC6 expression.14 Upregulated MUC2 and MUC5AC bind with EGFR and activate the Ras/Raf pathway, which in turn leads to cell proliferation. Notch-1 regulates MUC5AC synthesis in NCI-H292 human lung carcinoma cells through the ERK pathway (Fig. 3).44 MUC5AC gene expression is strongly repressed by dexamethasone treatment, possibly through the glucocorticoid receptor and two glucocorticoid response elements in the MUC5AC promoter.45,46 The extracellular matrix also regulates MUC5AC production in H292 human lung cancer cells. When H292 cells are grown on type-IV collagen, MUC5AC production is downregulated through an integrin/Srcdependent pathway.47 Conversely, another study has shown that laminin upregulates MUC5AC production by means of an Src-dependent pathway.47 Bai et al.48 have reported that leukotriene D4 regulates the expression of both MUC2 and MUC5AC in NCI-H292 cells through the transcription factor SP-1, which is required for efficient transcription. Also, microRNA-378 positively correlates with MUC5AC expression in lung cancer cells and it results in increased proliferation and migration (Fig. 3).49 CDX is an intestine-specific homeobox gene that also regulates mucin gene expression, particularly MUC2 and MUC6.50 Human lung adenocarcinoma xenografts express more MUC6 than squamous cell carcinoma xenografts, and its expression correlates with transcription factors CDX1 and CDX2.50 Similarly, Rossi et al. have also reported that CDX2 regulates MUC2 gene transcription in lung cancer.51 MUC7 expression in lung and trachea has been shown to be significantly upregulated in response to cigarette smoke extract in lung cancer cells.52 Similarly, combined treatment of cigarette
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
smoke extract and lipopolysaccharide resulted in synergistic upregulation of MUC7 transcripts in NSCLC cell lines.52
MUCIN EXPRESSION: CLINICOPATHOLOGICAL CORRELATION MUC1 Situ et al. observed that MUC1 was more frequently expressed in adenocarcinoma (86.3%) than squamous cell carcinoma (39.1%) and other subtypes of NSCLC (74.1%). In this study, MUC1 was an independent prognostic factor for overall survival and disease-free survival in patients with stage IB NSCLC.53 Demirag et al.54 have demonstrated that MUC1 is detected in adenosquamous carcinoma of the lung and strongly associated with disease progression. MUC1 is differentially expressed in smoke-exposed lung and lung cancer tissue.55,56 Khodarev et al.57 have identified 35 genes, induced by MUC1, that may be involved in lung and breast tumorigenesis. Taking this study further, MacDermed et al. identified MUC1-associated proliferation signature. This study demonstrated that MUC1-cytoplasmic domain (MUC1CD)-associated genes are involved in DNA damage response and tumor formation.58 Serum KL-6 glycoprotein classified as MUC1 is a sensitive biomarker for interstitial lung diseases.59 Shields60 observed a positive association between circulating levels of KL-6 and subsequent risk of lung cancer. MUC1 mRNA expression was observed in the peripheral blood of 80% (48 of 60) of NSCLC patients compared with 65% (13 of 20) of healthy controls. Furthermore, aberrant MUC1 expression on the stroma-facing cell surface or diffuse distribution in the cytoplasm (cytoplasmic pattern) correlated with a poor prognosis from lung cancer.61
MUC4 Multiple studies have shown that MUC4 expression significantly correlates with the histological type of lung carcinoma.4,6,36,37,62 Increased expression of MUC4 was observed in squamous cell carcinoma, particularly central areas of cancer cell nests and in squamous pearls, but was absent in less differentiated peripheral tumor cells.6 MUC4 mRNA overexpression was also observed in lung cancer carcinoma cells compared with normal epithelium.4 In an immunohistochemical and reverse transcription-polymerase chain reaction analysis of 29 tumor samples, 67% expressed MUC4 protein and 72% of samples had MUC4 mRNA overexpression.4 Lower disease-free interval was observed in patients expressing high levels of MUC4 (≥25% of neoplastic cells stained).38 A recent study demonstrated that MUC4 deficient lung cancer cells induced epithelial to mesenchymal transition process; decreased MUC4 expression was found in patients who had adenocarcinoma involving lymph nodes.63 Similarly, we found that elevated expression of MUC4 was correlated with decreased proliferative capacity of lung cancer cells.39 These contrasting observations suggest that the actual role of MUC4 in lung cancer is unclear. MUC4 expression was observed by immunohistochemistry in most subtypes of NSCLC; adenocarcinomas 81% (151 of 187), 78% (69 of 88) of squamous cell carcinoma, 75% (6
Mucins in Lung Cancer
of 8) adenosquamous carcinoma, and 55% (33 of 60) of large cell carcinoma.6 Furthermore, MUC4 expression distinguished lung adenocarcinoma from malignant mesothelioma.36 A study showed that patients with stage I (138 patients) and II lung adenocarcinoma (17 patients) expressed significantly high levels of MUC4. Increased expression of MUC4 correlated with shorter disease-free interval and decreased overall survival in early stage lung adenocarcinoma.38 However, two other studies have shown that MUC4 expression is associated with better lung cancer outcomes.6,39 Our recent studies indicated that MUC4 expression in the primary tumor was greater than that in corresponding lymph node metastases.39 These differences could be attributed to stages, types of patients, number of samples analyzed and methods utilized for the analysis of MUC4. Other mucins MUC5AC and MUC6 seem to be expressed only in mucin-filled glandular and cystic lung adenocarcinoma.64 MUC5AC expression has been shown to be higher in adenocarcinomas compared with squamous cell carcinomas.15 Yu et al. observed MUC5AC overexpression in 26.2% (16 of 61) of stage I/II NSCLC patients and noted a significant association between MUC5AC expression and gender, adenocarcinoma histology, and a nonsmoking history. MUC5AC expression appears to increase the risk of distant metastasis and decrease overall survival from lung cancer.65 MUC5AC expression has been shown to increase significantly during the progression from AAH through BAC (classified as “adenocarcinoma in situ” in the 2011 IASLC/ ATS/ERS classification of adenocarcinoma) to adenocarcinoma and alterations in MUC5AC expression were associated with dedifferentiation of bronchial epithelium.66 MUC5B and MUC5AC overexpressing tumors tended to relapse or metastasize postoperatively more often, compared with nonexpressing tumors.65 In summary, there are increasing data regarding the clinical utility of various mucins as potential biomarkers in lung adenocarcinoma. However, based on the available evidence, no definite conclusions can be drawn about their utility at this time and more studies evaluating the clinical utility are required at this time.
MUCINS AS THERAPEUTIC TARGETS Several approaches targeting MUC1 are currently in clinical trials for NSCLC. MUC1 has T-cell-specific antigenic epitopes that bind to HLA class I molecules. MUC1 has been explored as a target for immunotherapy using immunodominant peptides from variable number of tandem repeat region region.67 L-BLP25 (tecemotide) is an liposomal vaccine targeting MUC168, and has been shown to be effective and safe in NSCLC.23 In one study, 16 of 78 patients had a specific T-cell proliferative response against MUC1 induced by the L-BLP25 vaccine.69 MUC1 (60mer peptide from the tandem-repeat sequence) immunization generated T-lymphocyte-mediated cytotoxicity specific to MUC1,70 an activity independent of the MHC-I complex.71 A recent phase IIb trial randomized stage IIIb/IV NSCLC patients for treatment with either L-BLP25 or best supportive care, this study demonstrated that although there was no improvement in
Copyright © 2014 by the International Association for the Study of Lung Cancer
25
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
Lakshmanan et al.
overall survival in the entire cohort, the subgroup of patients with stage IIIb disease had a statistically significant (30.6 versus 13.3 months; HR, 0.548) improvement in median overall survival.69 Similar studies also reported that tecemotide prolonged survival in East Asian patients with stage III NSCLC.72 Unfortunately, two studies, a randomized placebo-controlled phase III study and a phase II study being conducted to identify a possible role for tecemotide in locally advanced NSCLC after completion of chemoradiation (NCT02049151 and NCT00828009) were recently discontinued by the sponsor, because of concerns regarding a lack of efficacy. TG4010 is another MUC1-targeted vaccine based on recombinant attenuated strain of vaccina virus expressing MUC1 and IL-2.73 A phase II study using a combination of TG4010 with cisplatin and vinorelbine resulted in improved outcomes in patients receiving first-line chemotherapy. The response rate in this study was 29.5%, median survival was 12.7 months and 1-year survival was 53%.73 A randomized study evaluating the combination of TG4010 with platinumbased chemotherapy is currently underway. MUC1-specific aptamer is made by pDNA/polyethylenimine/aptamer (pDNA/PEI/aptamer) complexes. The pDNA/ PEI/MUC1 aptamer complexes was designed for tumorspecific gene delivery tissue.74 Under-glycosylated forms of MUC1 in tumor cells can be used for developing therapeutic vaccine strategies and biomarkers for prognosis.75
CONCLUSION AND PERSPECTIVES Although mucins have not been well studied in lung cancer, they seem to play an important role in lung carcinogenesis. Due to their specific expression pattern and functional involvement in lung cancer, mucins may provide important diagnostic and therapeutic targets. It is possible that the interactions between various mucins may add another level of complexity in our understanding of their role in lung carcinogenesis. MUC1-targeted therapies are currently in clinical trials against lung cancer. Depolarized expression of MUC1 is associated with aggressiveness of lung cancer, and MUC1associated genes have been identified as possible prognostic and diagnostic markers in lung cancer. MUC1-associated proliferation signature may have utility for diagnosis and treatment of lung cancer. MUC5AC is overexpressed in lung cancer and it is associated with a poor prognosis. Although there are no current data, the role of the secretory mucin MUC5AC as a prognostic biomarker for lung cancer is worth investigating. More detailed studies are also needed to elucidate the role of mucins in lung cancers with different mutational background such as Kras, EGFR, and BRAF. Further studies are required to identify specific mucins that can serve as diagnostic or prognostic markers during the initiation and progression of lung cancer and to gain a better understanding of the unique role for each mucin and their interplay with each other during lung carcinogenesis.
ACKNOWLEDGMENT
The study is supported by a grant from the U.S. Department of Veterans Affairs.
26
REFERENCES 1. Van Klinken BJ, Einerhand AW, Buller HA, Dekker J. Strategic biochemical analysis of mucins. Anal Biochem 1998;265:103–116. 2. Kufe DW. Mucins in cancer: Function, prognosis and therapy. Nat Rev Cancer 2009;9:874–885. 3. Awaya H, Takeshima Y, Yamasaki M, Inai K. Expression of MUC1, MUC2, MUC5AC, and MUC6 in atypical adenomatous hyperplasia, bronchioloalveolar carcinoma, adenocarcinoma with mixed subtypes, and mucinous bronchioloalveolar carcinoma of the lung. Am J Clin Pathol 2004;121:644–653. 4. Hanaoka J, Kontani K, Sawai S, et al. Analysis of MUC4 mucin expression in lung carcinoma cells and its immunogenicity. Cancer 2001;92:2148–2157. 5. Kaira K, Okumura T, Nakagawa K, et al. MUC1 expression in pulmonary metastatic tumors: A comparison of primary lung cancer. Pathol Oncol Res 2012;18:439–447. 6. Kwon KY, Ro JY, Singhal N, et al. MUC4 expression in non-small cell lung carcinomas: Relationship to tumor histology and patient survival. Arch Pathol Lab Med 2007;131:593–598. 7. Hollingsworth MA, Swanson BJ. Mucins in cancer: Protection and control of the cell surface. Nat Rev Cancer 2004;4:45–60. 8. Gao J, McConnell MJ, Yu B, et al. MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion. Int J Oncol 2009;35:337–345. 9. Kim KC, Lillehoj EP. MUC1 mucin: A peacemaker in the lung. Am J Respir Cell Mol Biol 2008;39:644–647. 10. Lakshmanan I, Ponnusamy MP, Das S, et al. MUC16 induced rapid G2/M transition via interactions with JAK2 for increased proliferation and antiapoptosis in breast cancer cells. Oncogene 2012;31:805–817. 11. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9–29. 12. Rose MC. Mucins: Structure, function, and role in pulmonary diseases. Am J Physiol 1992;263:L413–L429. 13. Buisine MP, Devisme L, Copin MC, et al. Developmental mucin gene expression in the human respiratory tract. Am J Respir Cell Mol Biol 1999;20:209–218. 14. Perrais M, Pigny P, Copin MC, Aubert JP, Van Seuningen I. Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signalregulated kinase cascade and Sp1. J Biol Chem 2002;277:32258–32267. 15. Copin MC, Devisme L, Buisine MP, et al. From normal respiratory mucosa to epidermoid carcinoma: Expression of human mucin genes. Int J Cancer 2000;86:162–168. 16. Roy MG, Rahmani M, Hernandez JR, et al. Mucin production during prenatal and postnatal murine lung development. Am J Respir Cell Mol Biol 2011;44:755–760. 17. Hilkens J, Ligtenberg MJ, Vos HL, Litvinov SV. Cell membrane-associated mucins and their adhesion-modulating property. Trends Biochem Sci 1992;17:359–363. 18. Nath S, Mukherjee P. MUC1: A multifaceted oncoprotein with a key role in cancer progression. Trends Mol Med 2014;20:332–342. 19. Kaira K, Nakagawa K, Ohde Y, et al. Depolarized MUC1 expression is closely associated with hypoxic markers and poor outcome in resected non-small cell lung cancer. Int J Surg Pathol 2012;20:223–232. 20. Jarrard JA, Linnoila RI, Lee H, Steinberg SM, Witschi H, Szabo E. MUC1 is a novel marker for the type II pneumocyte lineage during lung carcinogenesis. Cancer Res 1998;58:5582–5589. 21. Guddo F, Giatromanolaki A, Patriarca C, et al. Depolarized expression of episialin (EMA, MUC1) in lung adenocarcinoma is associated with tumor progression. Anticancer Res 1998;18:1915–1920. 22. Nagai S, Takenaka K, Sonobe M, Ogawa E, Wada H, Tanaka F. A novel classification of MUC1 expression is correlated with tumor differentiation and postoperative prognosis in non-small cell lung cancer. J Thorac Oncol 2006;1:46–51. 23. Giatromanolaki A, Koukourakis MI, Sivridis E, et al. Coexpression of MUC1 glycoprotein with multiple angiogenic factors in non-small cell lung cancer suggests coactivation of angiogenic and migration pathways. Clin Cancer Res 2000;6:1917–1921. 24. Makiguchi Y, Hinoda Y, Imai K. Effect of MUC1 mucin, an anti-adhesion molecule, on tumor cell growth. Jpn J Cancer Res 1996;87:505–511. 25. Mikami Y, Hisatsune A, Tashiro T, Isohama Y, Katsuki H. Hypoxia enhances MUC1 expression in a lung adenocarcinoma cell line. Biochem Biophys Res Commun 2009;379:1060–1065. 26. Pochampalli MR, el Bejjani RM, Schroeder JA. MUC1 is a novel regulator of ErbB1 receptor trafficking. Oncogene 2007;26:1693–1701.
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
27. Li Y, Ren J, Yu W, et al. The epidermal growth factor receptor regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and beta-catenin. J Biol Chem 2001;276:35239–35242. 28. Bitler BG, Menzl I, Huerta CL, et al. Intracellular MUC1 peptides inhibit cancer progression. Clin Cancer Res 2009;15:100–109. 29. Klinge CM, Radde BN, Imbert-Fernandez Y, et al. Targeting the intracellular MUC1 C-terminal domain inhibits proliferation and estrogen receptor transcriptional activity in lung adenocarcinoma cells. Mol Cancer Ther 2011;10:2062–2071. 30. Ramasamy S, Duraisamy S, Barbashov S, Kawano T, Kharbanda S, Kufe D. The MUC1 and galectin-3 oncoproteins function in a microRNAdependent regulatory loop. Mol Cell 2007;27:992–1004. 31. Raina D, Kharbanda S, Kufe D. The MUC1 oncoprotein activates the antiapoptotic phosphoinositide 3-kinase/Akt and Bcl-xL pathways in rat 3Y1 fibroblasts. J Biol Chem 2004;279:20607–20612. 32. Raina D, Kosugi M, Ahmad R, et al. Dependence on the MUC1-C oncoprotein in non-small cell lung cancer cells. Mol Cancer Ther 2011;10:806–816. 33. Kuwahara I, Lillehoj EP, Hisatsune A, et al. Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter. Am J Physiol Lung Cell Mol Physiol 2005;289:L355–L362. 34. Molina-Pinelo S, Gutiérrez G, Pastor MD, et al. MicroRNA-dependent regulation of transcription in non-small cell lung cancer. PLoS One 2014;9:e90524. 35. Bansil R, Stanley E, LaMont JT. Mucin biophysics. Annu Rev Physiol 1995;57:635–657. 36. Llinares K, Escande F, Aubert S, et al. Diagnostic value of MUC4 immunostaining in distinguishing epithelial mesothelioma and lung adenocarcinoma. Mod Pathol 2004;17:150–157. 37. Karg A, Dinç ZA, Başok O, Uçvet A. MUC4 expression and its relation to ErbB2 expression, apoptosis, proliferation, differentiation, and tumor stage in non-small cell lung cancer (NSCLC). Pathol Res Pract 2006;202:577–583. 38. Tsutsumida H, Goto M, Kitajima S, et al. MUC4 expression correlates with poor prognosis in small-sized lung adenocarcinoma. Lung Cancer 2007;55:195–203. 39. Majhi PD, Lakshmanan I, Ponnusamy MP, et al. Pathobiological implications of MUC4 in non-small-cell lung cancer. J Thorac Oncol 2013;8:398–407. 40. Damera G, Xia B, Ancha HR, Sachdev GP. IL-9 modulated MUC4 gene and glycoprotein expression in airway epithelial cells. Biosci Rep 2006;26:55–67. 41. Yamada N, NishidaY, Tsutsumida H, et al. Promoter CpG methylation in cancer cells contributes to the regulation of MUC4. Br J Cancer 2009;100:344–351. 42. Barsky SH, Cameron R, Osann KE, Tomita D, Holmes EC. Rising incidence of bronchioloalveolar lung carcinoma and its unique clinicopathologic features. Cancer 1994;73:1163–1170. 43. Kim S, Lewis C, Nadel JA. CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells. J Immunol 2011;186:3392–3400. 44. Kang JH, Lee EH, Park SW, Chung IY. MUC5AC expression through bidirectional communication of Notch and epidermal growth factor receptor pathways. J Immunol 2011;187:222–229. 45. Chen Y, Watson AM, Williamson CD, et al. Glucocorticoid receptor and histone deacetylase-2 mediate dexamethasone-induced repression of MUC5AC gene expression. Am J Respir Cell Mol Biol 2012;47:637–644. 46. Chen Y, Zhao YH, Di YP, Wu R. Characterization of human mucin 5B gene expression in airway epithelium and the genomic clone of the amino-terminal and 5’-flanking region. Am J Respir Cell Mol Biol 2001;25:542–553. 47. Iwashita J, Yamamoto T, Sasaki Y, Abe T. MUC5AC production is downregulated in NCI-H292 lung cancer cells cultured on type-IV collagen. Mol Cell Biochem 2010;337:65–75. 48. Bai CH, Song SY, Kim YD. The inhibitory effect of the leukotriene receptor antagonist on leukotriene D4-induced MUC2/5AC gene expression and mucin secretion in human airway epithelial cells. Auris Nasus Larynx 2007;34:203–206. 49. Skrzypek K, Tertil M, Golda S, et al. Interplay between heme oxygenase-1 and miR-378 affects non-small cell lung carcinoma growth, vascularization, and metastasis. Antioxid Redox Signal 2013;19:644–660. 50. Hamamoto A, Abe Y, Nishi M, et al. Aberrant expression of the gastric mucin MUC6 in human pulmonary adenocarcinoma xenografts. Int J Oncol 2005;26:891–896. 51. Rossi G, Murer B, Cavazza A, et al. Primary mucinous (so-called colloid) carcinomas of the lung: A clinicopathologic and immunohistochemical
Mucins in Lung Cancer
study with special reference to CDX-2 homeobox gene and MUC2 expression. Am J Surg Pathol 2004;28:442–452. 52. Fan H, Bobek LA. Regulation of human MUC7 mucin gene expression by cigarette smoke extract or cigarette smoke and Pseudomonas aeruginosa lipopolysaccharide in human airway epithelial cells and in MUC7 transgenic mice. Open Respir Med J 2010;4:63–70. 53. Situ D, Wang J, Ma Y, et al. Expression and prognostic relevance of MUC1 in stage IB non-small cell lung cancer. Med Oncol 2011;28:S596–S604. 54. Demirag F, Cakir E, Bayiz H, Eren Yazici U. MUC1 and bcl-2 expression in preinvasive lesions and adenosquamous carcinoma of the lung. Acta Chir Belg 2013;113:19–24. 55. Powell E, Chow LQ. BLP-25 liposomal vaccine: A promising potential therapy in non-small-cell lung cancer. Expert Rev Respir Med 2008;2:37–45. 56. Spira A, Beane J, Shah V, et al. Effects of cigarette smoke on the human airway epithelial cell transcriptome. Proc Natl Acad Sci USA 2004;101:10143–10148. 57. Khodarev NN, Pitroda SP, Beckett MA, et al. MUC1-induced transcriptional programs associated with tumorigenesis predict outcome in breast and lung cancer. Cancer Res 2009;69:2833–2837. 58. MacDermed DM, Khodarev NN, Pitroda SP, et al. MUC1-associated proliferation signature predicts outcomes in lung adenocarcinoma patients. BMC Med Genomics 2010;3:16. 59. Ohnishi H, Yokoyama A, Kondo K, et al. Comparative study of KL-6, surfactant protein-A, surfactant protein-D, and monocyte chemoattractant protein-1 as serum markers for interstitial lung diseases. Am J Respir Crit Care Med 2002;165:378–381. 60. Shields PG. Molecular epidemiology of lung cancer. Ann Oncol 1999;10:S7–11. 61. Matsumura N, Yamamoto M, Aruga A, Takasaki K, Nakano M. Correlation between expression of MUC1 core protein and outcome after surgery in massforming intrahepatic cholangiocarcinoma. Cancer 2002;94:1770–1776. 62. Weed DT, Gomez-Fernandez C, Bonfante E, et al. MUC4 (sialomucin complex) expression in salivary gland tumors and squamous cell carcinoma of the upper aerodigestive tract. Otolaryngol Head Neck Surg 2001;124:127–141. 63. Gao L, Liu J, Zhang B, et al. Functional MUC4 suppress epithelialmesenchymal transition in lung adenocarcinoma metastasis. Tumour Biol 2001;35:1335–1341.. 64. Hishida T, Ishii G, Kodama T, et al. Centrally located adenocarcinoma with endobronchial polypoid growth: Clinicopathological analysis of five cases. Pathol Int 2011;61:73–79. 65. Yu CJ, Shih JY, Lee YC, Shun CT, Yuan A, Yang PC. Sialyl Lewis antigens: Association with MUC5AC protein and correlation with post-operative recurrence of non-small cell lung cancer. Lung Cancer 2005;47:59–67. 66. López-Ferrer A, Curull V, Barranco C, et al. Mucins as differentiation markers in bronchial epithelium. Squamous cell carcinoma and adenocarcinoma display similar expression patterns. Am J Respir Cell Mol Biol 2001;24:22–29. 67. Szabo E. MUC1 expression in lung cancer. Methods Mol Med 2003;74:251–258. 68. Sangha R, Butts C. L-BLP25: A peptide vaccine strategy in non small cell lung cancer. Clin Cancer Res 2007;13:s4652–s4654. 69. Butts C, Murray N, Maksymiuk A, et al. Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol 2005;23:6674–6681. 70. Brossart P, Heinrich KS, Stuhler G, et al. Identification of HLA-A2restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies. Blood 1999;93:4309–4317. 71. Sharpe JC, Abel PD, Gilbertson JA, et al. Modulated expression of human leucocyte antigen class I and class II determinants in hyperplastic and malignant human prostatic epithelium. Br J Urol 1994;74:609–616. 72. Wu YL, Park K, Soo RA, et al. INSPIRE: A phase III study of the BLP25 liposome vaccine (L-BLP25) in Asian patients with unresectable stage III non-small cell lung cancer. BMC Cancer 2011;11:430. 73. Ramlau R, Quoix E, Rolski J, et al. A phase II study of Tg4010 (MvaMuc1-Il2) in association with chemotherapy in patients with stage III/IV non-small cell lung cancer. J Thorac Oncol 2008;3:735–744. 74. Kurosaki T, Higuchi N, Kawakami S, et al. Self-assemble gene delivery system for molecular targeting using nucleic acid aptamer. Gene 2012;491:205–209. 75. Rahn JJ, Dabbagh L, Pasdar M, Hugh JC. The importance of MUC1 cellular localization in patients with breast carcinoma: An immunohistologic study of 71 patients and review of the literature. Cancer 2001;91:1973–1982.
Copyright © 2014 by the International Association for the Study of Lung Cancer
27
Mucins in Lung Cancer Diagnostic, Prognostic, and Therapeutic Implications Imayavaramban Lakshmanan, PhD,* Moorthy P. Ponnusamy, PhD,* Muzafar A. Macha, PhD,* Dhanya Haridas, PhD,* Prabin Dhangada Majhi, PhD,* Sukhwinder Kaur, PhD,* Maneesh Jain, PhD,* Surinder K. Batra, PhD,*†‡ and Apar Kishor Ganti, MD, MS§║
Abstract: Aberrant expression of mucins is associated with cancer development and metastasis. An overexpression of few mucins contributes to oncogenesis by enhancing cancer cell growth and providing constitutive survival signals. This review focuses on the importance of mucins both in the normal bronchial epithelial cells and the malignant tumors of the lung and their contribution in the diagnosis and prognosis of lung cancer patients. During lung cancer progression, mucins either alone or through their interaction with many receptor tyrosine kinases mediate cell signals for growth and survival of cancer cells. Also, stage-specific expression of certain mucins, like MUC1, is associated with poor prognosis from lung cancer. Thus, mucins are emerging as attractive targets for developing novel therapeutic approaches for lung cancer. Several strategies targeting mucin expression and function are currently being investigated to control lung cancer progression. Key Words: Lung cancer, Mucins, MUC1, MUC4, MUC5AC, MUC7, Mucin vaccines, L-BLP25, TG4010, Aptamer. (J Thorac Oncol. 2015;10: 19–27)
M
ucins are high molecular weight, heavily glycosylated proteins that are expressed in the epithelial cells of various organs. The presence of a variable number of tandem repeats in the extracellular domain distinguishes mucins from other glycoproteins. The number of tandem repeats is unique to each mucin and contains several proline, threonine, and serine (PTS) residues, but is usually devoid of cysteine residues. This mucin domain/PTS region provides the acceptors for relatively short O-linked glycans (2–20 monosaccharides per chain).1 The PTS region forms the densely packed filamentous carbohydrate-rich moiety that determines most of the biophysical properties of
*Department of Biochemistry and Molecular Biology, †Department of Pathology and Microbiology, ‡Eppley Institute for Research in Cancer and Allied Diseases, §Department of Internal Medicine, VA NebraskaWestern Iowa Health Care System, Omaha, Nebraska, and ║Division of Oncology–Hematology, Department of Internal Medicine, University of Nebraska Medical Center, Omaha,Nebraska. Disclosure: The authors declare no conflict of interest. Address for Correspondence: Apar Kishor Ganti, MD, MS, FACP, Division of Oncology–Hematology, Department of Internal Medicine, University of Nebraska Medical Center, 987680 Nebraska Medical Center, Omaha, NE 68198. E-mail: [email protected] DOI: 10.1097/JTO.0000000000000404 Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/15/1001-0019
mucins. Mucins are classified into two major groups: membranebound mucins and the secretory mucins (Fig. 1). There are 11 membrane bound mucins, namely, MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC20 and MUC21; and seven secreted mucins, MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC8, and MUC19.2 Mucins are expressed during embryonic stages of lung development and are involved in the normal development of the lung. Several reports suggest that mucins are overexpressed in non–small-cell lung cancer (NSCLC) and can be used as suitable biomarkers for identifying and monitoring the progression of lung cancer.3–6 Under normal conditions, mucins serve as a protective barrier for epithelial cells of various organs including lung.7 Deregulation of membrane-bound and secretory mucins is associated with cancer progression and metastasis by altering various signaling pathways.8–10 Of the various mucins, membrane-bound mucin MUC1 and secretory mucin MUC5AC seem to be predominantly found in NSCLC and are correlated with disease progression. Lung cancer is the most common cause of cancerrelated deaths in the United States.11 Mucins have not been extensively studied for their role in the pathogenesis of lung cancer. This may be due to the complexity of mucin biology and the existence of multiple mucins with differing functions within the cell. However, as our knowledge of mucins in other diseases is increasing, the role of various mucins in lung cancer too will be understood better. This review addresses the current knowledge about the expressional status and role of mucins in lung cancer cell, their significance as prognostic and diagnostic markers and their potential as therapeutic targets.
EXPRESSION AND FUNCTION OF MUCINS IN NORMAL LUNG EPITHELIUM Mucins secreted by the goblet cells in the respiratory epithelium form the mucosal coat of the apical epithelium and thereby protect the airways and alveoli from insults such as dehydration, pathogen invasions, and chemical agents.12 In a normal airway, expression of MUC1 is weak, and a diffuse signal is observed in the tracheobronchial and collecting duct epithelium. MUC1 expression is not observed in the submucosal glands and bronchioles.13 Weak MUC2 expression is observed in the entire respiratory epithelium; however, the basal pole of some goblet cells expresses MUC2 moderately.13 MUC2 is undetectable in
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
19
Lakshmanan et al.
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
FIGURE 1. Structure of membrane-bound and secretory type mucins and their domain. Schematic representation of the most differentially expressed membrane-bound (MUC1 and MUC4) and secretory mucins (MUC5AC) in lung cancer and their associated O-glycans: lung cancer is characterized by differential expression of MUC1 and de novo expression of MUC4 and MUC5AC. Domain structure of MUC1: The epitome of membrane-bound mucins, MUC1, is characterized by the presence of an N-terminal sequence, 20 tandem repeat domains (20–120 amino acid each), single self-cleavable sea-urchin sperm protein, enterokinase, and agrin (SEA) domain (two subunits of MUC1 are generated by auto-proteolysis at the SEA domain that, in turn, forms a stable noncovalent heterodimer), single spanning TM domain and 72 amino acid (aa) cytoplasmic tail (CT) that contains binding sites for various effector molecules including ERα, p53, GSK-3β, PKCδ, cSRC, and β-catenin. The MUC1 cytoplasmic tail is target of various oncogenic signaling pathways. Domain structure of MUC4: De novo-expressed transmembrane mucin MUC4 is putatively cleaved at Gly-Asp-Pro-His (GDPH) proteolytic site generating an extracellular subunit MUC4α and the membrane-tethered subunit MUC4β. MUC4α is characterized by a unique N-terminal sequence, three imperfect repeats, heavily O-glycosylated tandem repeat domain of 16 amino-acids, a cysteine-rich domain (CRD), a nidogen (NIDO) domain having similarity to the nidogen-epidermal growth factor (EGF) domain of ancestral nidogen proteins, eight invariant cysteine residues containing the adhesion-associated domain in MUC4 and other proteins (AMOP) domain. MUC4β is a growth factor-like subunit characterized by the presence of three EGF like domains, a vWD-domain having similarity to dimerization domains of the von Willebrand D (VWD) factor, a membrane-spanning transmembrane domain and a short cytoplasmic tail of 32 amino acids. Domain structure of MUC5AC: MUC5AC, a differentially expressed gel-forming mucin, has N- and C-terminal domains with high similarity to cystein-rich domain that is involved in disulfide-linked multimer formation. It includes disulfide-rich D-domains (D1, D2, D’, and D3) at the N-terminus and D4, B, C, and CK (vWF-like domain) at the C-terminus. The central region is characterized by CRDs (n = 17, aa = 110 having 10 cysteine residues). The first nine CRD are interspersed by threonine/serine/proline-rich domains (TSP) with no repetitive sequences, whereas Cys5 to Cys9 domains are interspersed with highly O-glycosylated TR domains (N = 4). Through its CRDs and CK domain, it forms disulfide-linked dimers and oligomers. Furthermore, its C-terminus has a GDPH autocatalytic proteolytic site.
the bronchioles and alveolar epithelial cells.14 In contrast, most of the respiratory epithelial cells including the basal, ciliated, and goblet cells as well as the collecting ducts express MUC4.15
20
Strong expression of MUC4 is found in the collecting ducts of the sub-mucosal glands and alveolar epithelial cells.14 Interestingly, MUC4 is one of the first mucins expressed in the early stages
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
of embryonic development in the lungs (as early as 6.5 week after gestation),13 and it is speculated to be involved in the differentiation of all respiratory epithelial cells.13 After 12 weeks of gestation, MUC4 is observed in the small bronchi and bronchioles of lungs (Table 1).13,15 Moderate expression of MUC5AC is initially observed at the 13th week of gestation in the epithelial buds of the segmental bronchi. MUC5AC is strongly expressed in the trachea and main bronchi, but no expression is seen in the bronchioles and smaller alveolar epithelial cells.14 MUC5AC is also expressed in the goblet cells of the surface epithelium and in the glandular ducts.14 MUC5B starts to appear after the 13th week of gestation and a weak expression is observed in the bronchial epithelium, mucous cells of the sub-mucosal glands, and glandular ducts.13,15 MUC5B is not expressed in the bronchioles and alveolar epithelial cells, but is strongly expressed in the collecting ducts and mucous cells of sub-mucosal glands.16 MUC7 transcript is detected only in the serous cells of some sub-mucosal glands.15 Other mucins such as MUC3 and MUC6 are not expressed in the normal respiratory mucosa.15 In summary, mucins exhibit distinct spatiotemporal expression pattern in different regions of the respiratory epithelium and during lung development (Table 1).
MUCINS IN LUNG CANCER MUC1 In normal epithelial cells, MUC1 expression is restricted to the luminal side of the cells.17 However, during progression of lung cancer, there is a loss of cell polarity with resulting MUC1 expression over the entire cell surface.18 MUC1 expression has been classified into three types, high-grade polarized expression (HP), low-grade polarized expression (LP), and depolarized expression (DP). HP is defined as having polarized expression of MUC1 in greater than 50% tumor cells and less than 10% cells showing depolarized expression of MUC1. LP is less than
Mucins in Lung Cancer
50% of cells with polarized expression of MUC1 and less than 10% of depolarized expression of MUC1, whereas DP is considered as depolarized MUC1 expression in greater than 10% of cancer cells.5,19 Based on this classification, HP of MUC1 was seen in well-differentiated adenocarcinoma, whereas depolarized expression of MUC1 correlated with increasing stage and lymph node metastasis.20,21 Depolarization of MUC1 expression extends from the apex and moves across the entire surface thereby preventing cell–cell and cell–matrix interaction, thereby resulting in increased invasion into adjacent tissues. Differential polarized expression of MUC1 was also observed between primary and pulmonary metastatic tumors. HP and DP of MUC1 were significantly higher in primary lung cancer tissues than in that of metastatic lesions.5 Increased depolarized expression of MUC1 also correlated with progression of atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC; adenocarcinoma with lepidic pattern in the new International Association for the Study of Lung Cancer [IASLC] classification).3 The clinicopathological significance of MUC1 was analyzed in 126 patients with primary lung cancer and 147 patients with metastatic tumors in the lung. The (18)F-FDG uptake was higher in patients with depolarized expression (DP) as compared with namely HP or LP. HP and DP pattern of MUC1 was seen in a higher proportion of patients with primary lung tumors as compared with metastatic lesions but LP of MUC1 was detected in a higher proportion of patients with metastatic lesions.5 The study suggested that depolarized expression of MUC1 was associated with poor prognosis. These findings suggest that differential distribution of MUC1 plays a significant role in aggressiveness of lung cancer and metastasis. MUC1 signaling and its role in aggressiveness of lung cancer cells MUC1 overexpression induces several downstream signaling pathways that are closely associated with poor clinical
TABLE 1. Mucins Expression in Normal Bronchial Cells and Carcinomas Mucins
Expression in Normal Respiratory Tract
MUC1
Trachea bronchial epithelium, collecting ducts, submucosal gland and bronchioles13
MUC4
Goblet cells, collecting duct, and alveolar epithelial cells13,15
MUC2 MUC5AC MUC5B
Respiratory epithelium, goblet cells13 Surface epithelium of goblet cells, trachea, and main bronchi13,14 Bronchioles and alveolar epithelial cells, collecting duct and submucosal gland13,15 Serous cells of submucosal gland.13,15
MUC7
Expression in Preinvasive and Invasive Lesions Preinvasive:54 All preinvasive lesions: 77% (34 of 44) Squamous dysplasia: 83.3% (20 of 24) Bronchiolar columnar cell dysplasia: 83.3% (5 of 6) Basal cell dysplasia: 66.6% (4 of 6) Bronchial epithelial dysplasia with transitional differentiation: 66.6% (2 of 3) Columnar cell dysplasia: 66.6% (4 of 6) Invasive:54 Squamous component: 96.7% (30 of 31) Adenocarcinoma components: 87% (27 of 31) Adenocarcinomas: 81% (151 of 187)6 Squamous cell carcinomas: 78% (69 of 88) Adenosquamous carcinomas: 75% (6 of 8) Large cell carcinomas: 55% (33 of 60) No data Stage I NSCLC: 16 of 61 (26.2%)74 No data No data
BCCD, bronchiolar columnar cell dysplasia; BCD, basal cell dysplasia; BEDT, bronchial epithelial dysplasia with transitional differentiation; CCD, columnar cell dysplasia; NSCLC, non–small-cell lung cancer.
Copyright © 2014 by the International Association for the Study of Lung Cancer
21
Lakshmanan et al.
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
outcomes.22 Overexpression of MUC1 favors angiogenesis by upregulating vascular endothelial growth factor (VEGF) expression through activation of the Akt and ERK signaling pathways. Giatromanolaki et al.23 demonstrated that highly vascularized NSCLC tumors had high MUC1 expression, and this appeared to correlate with VEGF expression. MUC1 is also involved in the inactivation of integrin-mediated cell-to-matrix adhesion and E-cadherin mediated cell-to-cell adhesion.24 MUC1 knockdown led to sensitization of NSCLC cell lines to cisplatin treatment and elevated apoptosis due to reduced Bcl-XL activity.25 MUC1 has been shown to interact with EGFR and β-catenin through its cytoplasmic domain and promote cancer cell proliferation.26 MUC1 cytoplasmic domain contains 72 amino acids; a 15 amino acid sequence is responsible for interaction with EGFR (YEKV) and β catenin (SAGNGGSSLS).27 Bitler et al.28 synthesized a MUC1 inhibitory peptide to block these interactions along protein
transduction domain PTD4 for enhancing the cellular uptake. MUC1 inhibition in lung cancer cells led to decreased proliferation by affecting transcription of the estradiol-activated reporter genes, endogenous cyclin D1, and nuclear respiratory factor-1.29 MUC1 C-terminal (MUC1-C) subunit forms a complex with EGFR by interacting with Galectin-3.30 In addition, the MUC1-C acts as a substrate for c-Src phosphorylation in lung cancer cells.27 Recently, MUC1-C was reported to be aberrantly expressed in NSCLC and through its association with p85 subunit of PI3K was found to enhance the Akt signaling pathway.31 MUC1 undergoes oligomerization through its CysGln-Cys (CQC) motif present on its cytoplasmic domain and promotes cancer cell proliferation. The MUC1 inhibitor (GO-201) inhibits MUC1 oligomerization by binding to the cytoplasmic domain and reduces MUC1-mediated cell proliferation (Fig. 2).32 In addition, other MUC1-specific
FIGURE 2. Differential signaling of mucins in lung cancer pathogenesis. Overexpression of MUC1 activates various signaling pathways for lung cancer cell proliferation through PI3K and Akt activity, MUC1 also participates in angiogenesis through regulation VEGF expression. MUC1 and EGFR signaling also promote lung cancer cell proliferation through Src signaling pathways. Similarly, secretory mucin MUC5AC promotes lung cancer cell proliferation by alterating Ras signaling pathways and cytokines. Several inhibitors are available to inhibit the lung cancer cell proliferation either by directly targeting MUC1 by L-BLP25, G0203, or by Mucins downstream target such mTor and ERK1/2 and Akt. EGF, epidermal growth factor; mTOR, mammalian target of rapamycin; ER-α and β, estrogen receptor α and β; VEGF, vascular endothelial growth factor; SRC-v-src, aviansarcoma.
22
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
inhibitors such as GO-202 and 203 were also associated with an impaired proliferation of lung adenocarcinoma cells.29 Blocking the oligomerization of MUC1-C by G0-203 inhibited NSCLC cell growth and survival by preventing the interaction between MUC1-C and PI3K-p85. This resulted in the inhibition of Akt phosphorylation and mammalian target of rapamycin activity (Fig. 2).32
Mucins in Lung Cancer
MUC1 gene can be regulated by various signaling molecules and transcription factors in the airway epithelial cells and lung cancer cells. STAT3 is highly overexpressed in NSCLC and its knockdown significantly downregulates MUC1 expression, indicating that STAT3 regulates MUC1 gene expression in lung cancer cells (Fig. 3).8 Mikami et al.25 showed that hypoxia induced MUC1 expression in lung
FIGURE 3. Regulation of mucin genes through various signaling pathways in lung cancer cells: The transcription factor STAT3 regulates MUC1 gene expression in lung cancer cells under hypoxia condition through HIF1-α.8 Further MUC1gene is also regulated by microRNA149 in lung cancer cells.34 TNF-α–converting enzyme (TACE) also regulates both MUC1 and MUC5AC in lung cancer cells. For MUC1 gene expression, neutrophil elastase may activate Protein kinase C δ for expression of dual oxidase 1 (Duox1) that leads to reactive oxygen species production for TACE activation. Then TACE induced MUC1 gene expression through TNF-α/TNF-α-Receptor/ERK1/2/SP-1 transcription factor (Pathway A). For MUC5AC gene expression, chemokine (C–C motif) ligand (CCL20) binds with GPCR complex through EGFR activation. Then TACE induce the MUC5AC gene expression through ERK1/2 signaling (Pathway B). Notch1 upregulates EGFR expression that leads to MUC5AC gene expression through ERK signaling.44 (Pathway C.) In addition, MUC5AC gene can also be regulated by microRNA-378 in lung cancer and its expression positively correlates with MUC5AC gene expression.49 MUC2 and MUC6 genes may be regulated by CDX transcription factors in lung cancer cells. Collectively EGFR and its downstream signaling molecule ERK1/2 are necessary for mucin gene expression in lung cancer cells. STAT3, signal transducer and activator of transcription 3; EGFR, epidermal growth factor receptor 1; TGF-α, transforming growth factor-α; ERK1/2, extracellular signal-regulated protein kinases 1 and 2; HIF1α, hypoxiainducible factor 1-α; CCL20-Chemokine (C–C motif) ligand 20; GPCR, G protein-coupled receptors; TACE-TGF-α, tumor necrosis factor-α–converting enzyme, CDX1 and 2, caudal type homeobox 1&2. Similarly, MUC2 and MUC6 genes can be regulated by CDX molecules as well as SP-1. Copyright © 2014 by the International Association for the Study of Lung Cancer
23
Lakshmanan et al.
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
adenocarcinoma cells by means of HIF-1α, and was partially responsible for lung cancer progression. Neutrophil elastase regulates MUC1 mucin gene expression through Sp-1 transcription factor in lung cancer cells. Specific binding of Sp-1 on the MUC1 gene promoter between -99/-90 induced gene transcription (Fig. 3).33 Similarly, the same group has elucidated the detailed signaling mechanism of Sp-1-mediated MUC1 gene expression, e.g., neutrophil elastase can activate protein kinase δ and dual oxidase 1 (Duox1) that leads to reactive oxygen species production for TNFα-converting enzyme (TACE). TACE, in turn, activates TNFα and its receptor TNFR and results in MUC1 gene expression through MEK1/2, ERK1/2, and Sp-1 (Fig. 3). This study suggests that the anti-inflammatory role of MUC1 is controlled by inflammatory mediators, such as neutrophil elastase and TNFα.33 In addition, microRNA 149 has shown to repress MUC1 expression in lung adenocarcinoma.34 Thus, MUC1 is overexpressed in lung carcinoma and its expression is tightly regulated by a number of inflammatory regulators (Fig. 3).
MUC4 MUC4 is aberrantly overexpressed in many tumors including lung carcinoma.35 Membrane-bound MUC4 is widely expressed in NSCLC, but its expression varies between different histologic subtypes. Adenocarcinoma cells express more MUC4 than squamous cell carcinoma and large cell carcinoma cells.6 MUC4 expression was detected in the pleural infiltration zone and has been shown to be required for lung adenocarcinoma development.36 Karg et al.37 reported a significant association of MUC4 expression with HER2/ERBB2 that resulted in apoptosis and differentiation rather than lung cancer cell proliferation. In contrast, other studies have demonstrated that MUC4 expression was unrelated to MUC1, HER2, and p27 expression, thereby suggesting that MUC4 can be used as an independent prognostic marker for lung adenocarcinoma.38 Recently, we demonstrated that MUC4 expression was associated with decreased proliferation, motility and invasion of lung cancer cells by attenuating the activation of Akt, Cyclin D1, and Cyclin E39 (Fig. 2). In lungs, MUC4 gene expression is controlled by IL-9 in a time- and dose-dependent manner; Jak3 appears to be involved in IL9-mediated MUC4 gene expression (Fig. 3).40 MUC4 seems to also be controlled by an epigenetic mechanism. When the DNA methylation status of 94 CpG sites from −3622 to +29 was analyzed using a MassARRAY analysis, the MUC4 expressing H292 lung cancer cell line had low methylation levels (near the transcriptional start site) and hypomethylation at the CpG sites 108 to 112 was associated with MUC4 expression.41 Similarly, when non-MUC4 expressing A427 lung cancer cells were treated with hypomethylating agents, 5-aza-2′-deoxycytidine and trichostatin A, there was an increase in the MUC4 mRNA expression suggesting that methylation status may regulate MUC4 expression. Secretory mucins MUC5AC, MUC5B, MUC2, MUC6, and MUC7 Secretory mucins protect tumor cells from exogenous cytotoxicity or genotoxicity and contribute to their
24
aggressiveness. Mucinous adenocarcinoma, a more aggressive subtype of lung adenocarcinoma, has a high expression of MUC2, MUC5AC, and MUC6. The aggressiveness of the tumor may be dependent on the mucins it secretes.42 Of all the mucins in airway epithelium, the regulation of MUC5AC is most well studied. Human MUC5AC expression is regulated by chemokine (C–C motif) ligand (CCL20)/ GPCR complex by EGFR activation by means of metalloprotease TACE.43 A recent study has elucidated two different mechanisms of EGFR-mediated MUC5AC synthesis and secretion in H292 lung cancer cells: (1) EGFR phosphorylation upon binding to TGF-α results in the production of MUC5AC and CCL20 by means of ERK activation, and (2) EGFR phosphorylation is directly activated by TACE which cleaves the proligands specific for EGFR43 (Fig. 3). In atypical adenomatous hyperplasia (AAH) and BAC with mixed subtypes (classified as “adenocarcinoma, predominantly invasive with some nonmucinous lepidic component” in the 2011 IASLC/ATS/ERS classification of adenocarcinoma), cells express high levels of secretory mucins such as MUC2, MUC5AC, and MUC6. The expression of MUC2 and MUC5AC are controlled by epidermal growth factor family and mitogen-activated protein kinase (MAPK) cascade.14 MUC2 expression was also associated with wild type p53, but not mutant p53. Treatment of H292 lung cancer cells with EGF and TNF-α resulted in increased expression of MUC2 and MUC5AC but there was no change in MUC5B and MUC6 expression.14 Upregulated MUC2 and MUC5AC bind with EGFR and activate the Ras/Raf pathway, which in turn leads to cell proliferation. Notch-1 regulates MUC5AC synthesis in NCI-H292 human lung carcinoma cells through the ERK pathway (Fig. 3).44 MUC5AC gene expression is strongly repressed by dexamethasone treatment, possibly through the glucocorticoid receptor and two glucocorticoid response elements in the MUC5AC promoter.45,46 The extracellular matrix also regulates MUC5AC production in H292 human lung cancer cells. When H292 cells are grown on type-IV collagen, MUC5AC production is downregulated through an integrin/Srcdependent pathway.47 Conversely, another study has shown that laminin upregulates MUC5AC production by means of an Src-dependent pathway.47 Bai et al.48 have reported that leukotriene D4 regulates the expression of both MUC2 and MUC5AC in NCI-H292 cells through the transcription factor SP-1, which is required for efficient transcription. Also, microRNA-378 positively correlates with MUC5AC expression in lung cancer cells and it results in increased proliferation and migration (Fig. 3).49 CDX is an intestine-specific homeobox gene that also regulates mucin gene expression, particularly MUC2 and MUC6.50 Human lung adenocarcinoma xenografts express more MUC6 than squamous cell carcinoma xenografts, and its expression correlates with transcription factors CDX1 and CDX2.50 Similarly, Rossi et al. have also reported that CDX2 regulates MUC2 gene transcription in lung cancer.51 MUC7 expression in lung and trachea has been shown to be significantly upregulated in response to cigarette smoke extract in lung cancer cells.52 Similarly, combined treatment of cigarette
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
smoke extract and lipopolysaccharide resulted in synergistic upregulation of MUC7 transcripts in NSCLC cell lines.52
MUCIN EXPRESSION: CLINICOPATHOLOGICAL CORRELATION MUC1 Situ et al. observed that MUC1 was more frequently expressed in adenocarcinoma (86.3%) than squamous cell carcinoma (39.1%) and other subtypes of NSCLC (74.1%). In this study, MUC1 was an independent prognostic factor for overall survival and disease-free survival in patients with stage IB NSCLC.53 Demirag et al.54 have demonstrated that MUC1 is detected in adenosquamous carcinoma of the lung and strongly associated with disease progression. MUC1 is differentially expressed in smoke-exposed lung and lung cancer tissue.55,56 Khodarev et al.57 have identified 35 genes, induced by MUC1, that may be involved in lung and breast tumorigenesis. Taking this study further, MacDermed et al. identified MUC1-associated proliferation signature. This study demonstrated that MUC1-cytoplasmic domain (MUC1CD)-associated genes are involved in DNA damage response and tumor formation.58 Serum KL-6 glycoprotein classified as MUC1 is a sensitive biomarker for interstitial lung diseases.59 Shields60 observed a positive association between circulating levels of KL-6 and subsequent risk of lung cancer. MUC1 mRNA expression was observed in the peripheral blood of 80% (48 of 60) of NSCLC patients compared with 65% (13 of 20) of healthy controls. Furthermore, aberrant MUC1 expression on the stroma-facing cell surface or diffuse distribution in the cytoplasm (cytoplasmic pattern) correlated with a poor prognosis from lung cancer.61
MUC4 Multiple studies have shown that MUC4 expression significantly correlates with the histological type of lung carcinoma.4,6,36,37,62 Increased expression of MUC4 was observed in squamous cell carcinoma, particularly central areas of cancer cell nests and in squamous pearls, but was absent in less differentiated peripheral tumor cells.6 MUC4 mRNA overexpression was also observed in lung cancer carcinoma cells compared with normal epithelium.4 In an immunohistochemical and reverse transcription-polymerase chain reaction analysis of 29 tumor samples, 67% expressed MUC4 protein and 72% of samples had MUC4 mRNA overexpression.4 Lower disease-free interval was observed in patients expressing high levels of MUC4 (≥25% of neoplastic cells stained).38 A recent study demonstrated that MUC4 deficient lung cancer cells induced epithelial to mesenchymal transition process; decreased MUC4 expression was found in patients who had adenocarcinoma involving lymph nodes.63 Similarly, we found that elevated expression of MUC4 was correlated with decreased proliferative capacity of lung cancer cells.39 These contrasting observations suggest that the actual role of MUC4 in lung cancer is unclear. MUC4 expression was observed by immunohistochemistry in most subtypes of NSCLC; adenocarcinomas 81% (151 of 187), 78% (69 of 88) of squamous cell carcinoma, 75% (6
Mucins in Lung Cancer
of 8) adenosquamous carcinoma, and 55% (33 of 60) of large cell carcinoma.6 Furthermore, MUC4 expression distinguished lung adenocarcinoma from malignant mesothelioma.36 A study showed that patients with stage I (138 patients) and II lung adenocarcinoma (17 patients) expressed significantly high levels of MUC4. Increased expression of MUC4 correlated with shorter disease-free interval and decreased overall survival in early stage lung adenocarcinoma.38 However, two other studies have shown that MUC4 expression is associated with better lung cancer outcomes.6,39 Our recent studies indicated that MUC4 expression in the primary tumor was greater than that in corresponding lymph node metastases.39 These differences could be attributed to stages, types of patients, number of samples analyzed and methods utilized for the analysis of MUC4. Other mucins MUC5AC and MUC6 seem to be expressed only in mucin-filled glandular and cystic lung adenocarcinoma.64 MUC5AC expression has been shown to be higher in adenocarcinomas compared with squamous cell carcinomas.15 Yu et al. observed MUC5AC overexpression in 26.2% (16 of 61) of stage I/II NSCLC patients and noted a significant association between MUC5AC expression and gender, adenocarcinoma histology, and a nonsmoking history. MUC5AC expression appears to increase the risk of distant metastasis and decrease overall survival from lung cancer.65 MUC5AC expression has been shown to increase significantly during the progression from AAH through BAC (classified as “adenocarcinoma in situ” in the 2011 IASLC/ ATS/ERS classification of adenocarcinoma) to adenocarcinoma and alterations in MUC5AC expression were associated with dedifferentiation of bronchial epithelium.66 MUC5B and MUC5AC overexpressing tumors tended to relapse or metastasize postoperatively more often, compared with nonexpressing tumors.65 In summary, there are increasing data regarding the clinical utility of various mucins as potential biomarkers in lung adenocarcinoma. However, based on the available evidence, no definite conclusions can be drawn about their utility at this time and more studies evaluating the clinical utility are required at this time.
MUCINS AS THERAPEUTIC TARGETS Several approaches targeting MUC1 are currently in clinical trials for NSCLC. MUC1 has T-cell-specific antigenic epitopes that bind to HLA class I molecules. MUC1 has been explored as a target for immunotherapy using immunodominant peptides from variable number of tandem repeat region region.67 L-BLP25 (tecemotide) is an liposomal vaccine targeting MUC168, and has been shown to be effective and safe in NSCLC.23 In one study, 16 of 78 patients had a specific T-cell proliferative response against MUC1 induced by the L-BLP25 vaccine.69 MUC1 (60mer peptide from the tandem-repeat sequence) immunization generated T-lymphocyte-mediated cytotoxicity specific to MUC1,70 an activity independent of the MHC-I complex.71 A recent phase IIb trial randomized stage IIIb/IV NSCLC patients for treatment with either L-BLP25 or best supportive care, this study demonstrated that although there was no improvement in
Copyright © 2014 by the International Association for the Study of Lung Cancer
25
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
Lakshmanan et al.
overall survival in the entire cohort, the subgroup of patients with stage IIIb disease had a statistically significant (30.6 versus 13.3 months; HR, 0.548) improvement in median overall survival.69 Similar studies also reported that tecemotide prolonged survival in East Asian patients with stage III NSCLC.72 Unfortunately, two studies, a randomized placebo-controlled phase III study and a phase II study being conducted to identify a possible role for tecemotide in locally advanced NSCLC after completion of chemoradiation (NCT02049151 and NCT00828009) were recently discontinued by the sponsor, because of concerns regarding a lack of efficacy. TG4010 is another MUC1-targeted vaccine based on recombinant attenuated strain of vaccina virus expressing MUC1 and IL-2.73 A phase II study using a combination of TG4010 with cisplatin and vinorelbine resulted in improved outcomes in patients receiving first-line chemotherapy. The response rate in this study was 29.5%, median survival was 12.7 months and 1-year survival was 53%.73 A randomized study evaluating the combination of TG4010 with platinumbased chemotherapy is currently underway. MUC1-specific aptamer is made by pDNA/polyethylenimine/aptamer (pDNA/PEI/aptamer) complexes. The pDNA/ PEI/MUC1 aptamer complexes was designed for tumorspecific gene delivery tissue.74 Under-glycosylated forms of MUC1 in tumor cells can be used for developing therapeutic vaccine strategies and biomarkers for prognosis.75
CONCLUSION AND PERSPECTIVES Although mucins have not been well studied in lung cancer, they seem to play an important role in lung carcinogenesis. Due to their specific expression pattern and functional involvement in lung cancer, mucins may provide important diagnostic and therapeutic targets. It is possible that the interactions between various mucins may add another level of complexity in our understanding of their role in lung carcinogenesis. MUC1-targeted therapies are currently in clinical trials against lung cancer. Depolarized expression of MUC1 is associated with aggressiveness of lung cancer, and MUC1associated genes have been identified as possible prognostic and diagnostic markers in lung cancer. MUC1-associated proliferation signature may have utility for diagnosis and treatment of lung cancer. MUC5AC is overexpressed in lung cancer and it is associated with a poor prognosis. Although there are no current data, the role of the secretory mucin MUC5AC as a prognostic biomarker for lung cancer is worth investigating. More detailed studies are also needed to elucidate the role of mucins in lung cancers with different mutational background such as Kras, EGFR, and BRAF. Further studies are required to identify specific mucins that can serve as diagnostic or prognostic markers during the initiation and progression of lung cancer and to gain a better understanding of the unique role for each mucin and their interplay with each other during lung carcinogenesis.
ACKNOWLEDGMENT
The study is supported by a grant from the U.S. Department of Veterans Affairs.
26
REFERENCES 1. Van Klinken BJ, Einerhand AW, Buller HA, Dekker J. Strategic biochemical analysis of mucins. Anal Biochem 1998;265:103–116. 2. Kufe DW. Mucins in cancer: Function, prognosis and therapy. Nat Rev Cancer 2009;9:874–885. 3. Awaya H, Takeshima Y, Yamasaki M, Inai K. Expression of MUC1, MUC2, MUC5AC, and MUC6 in atypical adenomatous hyperplasia, bronchioloalveolar carcinoma, adenocarcinoma with mixed subtypes, and mucinous bronchioloalveolar carcinoma of the lung. Am J Clin Pathol 2004;121:644–653. 4. Hanaoka J, Kontani K, Sawai S, et al. Analysis of MUC4 mucin expression in lung carcinoma cells and its immunogenicity. Cancer 2001;92:2148–2157. 5. Kaira K, Okumura T, Nakagawa K, et al. MUC1 expression in pulmonary metastatic tumors: A comparison of primary lung cancer. Pathol Oncol Res 2012;18:439–447. 6. Kwon KY, Ro JY, Singhal N, et al. MUC4 expression in non-small cell lung carcinomas: Relationship to tumor histology and patient survival. Arch Pathol Lab Med 2007;131:593–598. 7. Hollingsworth MA, Swanson BJ. Mucins in cancer: Protection and control of the cell surface. Nat Rev Cancer 2004;4:45–60. 8. Gao J, McConnell MJ, Yu B, et al. MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion. Int J Oncol 2009;35:337–345. 9. Kim KC, Lillehoj EP. MUC1 mucin: A peacemaker in the lung. Am J Respir Cell Mol Biol 2008;39:644–647. 10. Lakshmanan I, Ponnusamy MP, Das S, et al. MUC16 induced rapid G2/M transition via interactions with JAK2 for increased proliferation and antiapoptosis in breast cancer cells. Oncogene 2012;31:805–817. 11. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9–29. 12. Rose MC. Mucins: Structure, function, and role in pulmonary diseases. Am J Physiol 1992;263:L413–L429. 13. Buisine MP, Devisme L, Copin MC, et al. Developmental mucin gene expression in the human respiratory tract. Am J Respir Cell Mol Biol 1999;20:209–218. 14. Perrais M, Pigny P, Copin MC, Aubert JP, Van Seuningen I. Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signalregulated kinase cascade and Sp1. J Biol Chem 2002;277:32258–32267. 15. Copin MC, Devisme L, Buisine MP, et al. From normal respiratory mucosa to epidermoid carcinoma: Expression of human mucin genes. Int J Cancer 2000;86:162–168. 16. Roy MG, Rahmani M, Hernandez JR, et al. Mucin production during prenatal and postnatal murine lung development. Am J Respir Cell Mol Biol 2011;44:755–760. 17. Hilkens J, Ligtenberg MJ, Vos HL, Litvinov SV. Cell membrane-associated mucins and their adhesion-modulating property. Trends Biochem Sci 1992;17:359–363. 18. Nath S, Mukherjee P. MUC1: A multifaceted oncoprotein with a key role in cancer progression. Trends Mol Med 2014;20:332–342. 19. Kaira K, Nakagawa K, Ohde Y, et al. Depolarized MUC1 expression is closely associated with hypoxic markers and poor outcome in resected non-small cell lung cancer. Int J Surg Pathol 2012;20:223–232. 20. Jarrard JA, Linnoila RI, Lee H, Steinberg SM, Witschi H, Szabo E. MUC1 is a novel marker for the type II pneumocyte lineage during lung carcinogenesis. Cancer Res 1998;58:5582–5589. 21. Guddo F, Giatromanolaki A, Patriarca C, et al. Depolarized expression of episialin (EMA, MUC1) in lung adenocarcinoma is associated with tumor progression. Anticancer Res 1998;18:1915–1920. 22. Nagai S, Takenaka K, Sonobe M, Ogawa E, Wada H, Tanaka F. A novel classification of MUC1 expression is correlated with tumor differentiation and postoperative prognosis in non-small cell lung cancer. J Thorac Oncol 2006;1:46–51. 23. Giatromanolaki A, Koukourakis MI, Sivridis E, et al. Coexpression of MUC1 glycoprotein with multiple angiogenic factors in non-small cell lung cancer suggests coactivation of angiogenic and migration pathways. Clin Cancer Res 2000;6:1917–1921. 24. Makiguchi Y, Hinoda Y, Imai K. Effect of MUC1 mucin, an anti-adhesion molecule, on tumor cell growth. Jpn J Cancer Res 1996;87:505–511. 25. Mikami Y, Hisatsune A, Tashiro T, Isohama Y, Katsuki H. Hypoxia enhances MUC1 expression in a lung adenocarcinoma cell line. Biochem Biophys Res Commun 2009;379:1060–1065. 26. Pochampalli MR, el Bejjani RM, Schroeder JA. MUC1 is a novel regulator of ErbB1 receptor trafficking. Oncogene 2007;26:1693–1701.
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 1, January 2015
27. Li Y, Ren J, Yu W, et al. The epidermal growth factor receptor regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and beta-catenin. J Biol Chem 2001;276:35239–35242. 28. Bitler BG, Menzl I, Huerta CL, et al. Intracellular MUC1 peptides inhibit cancer progression. Clin Cancer Res 2009;15:100–109. 29. Klinge CM, Radde BN, Imbert-Fernandez Y, et al. Targeting the intracellular MUC1 C-terminal domain inhibits proliferation and estrogen receptor transcriptional activity in lung adenocarcinoma cells. Mol Cancer Ther 2011;10:2062–2071. 30. Ramasamy S, Duraisamy S, Barbashov S, Kawano T, Kharbanda S, Kufe D. The MUC1 and galectin-3 oncoproteins function in a microRNAdependent regulatory loop. Mol Cell 2007;27:992–1004. 31. Raina D, Kharbanda S, Kufe D. The MUC1 oncoprotein activates the antiapoptotic phosphoinositide 3-kinase/Akt and Bcl-xL pathways in rat 3Y1 fibroblasts. J Biol Chem 2004;279:20607–20612. 32. Raina D, Kosugi M, Ahmad R, et al. Dependence on the MUC1-C oncoprotein in non-small cell lung cancer cells. Mol Cancer Ther 2011;10:806–816. 33. Kuwahara I, Lillehoj EP, Hisatsune A, et al. Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter. Am J Physiol Lung Cell Mol Physiol 2005;289:L355–L362. 34. Molina-Pinelo S, Gutiérrez G, Pastor MD, et al. MicroRNA-dependent regulation of transcription in non-small cell lung cancer. PLoS One 2014;9:e90524. 35. Bansil R, Stanley E, LaMont JT. Mucin biophysics. Annu Rev Physiol 1995;57:635–657. 36. Llinares K, Escande F, Aubert S, et al. Diagnostic value of MUC4 immunostaining in distinguishing epithelial mesothelioma and lung adenocarcinoma. Mod Pathol 2004;17:150–157. 37. Karg A, Dinç ZA, Başok O, Uçvet A. MUC4 expression and its relation to ErbB2 expression, apoptosis, proliferation, differentiation, and tumor stage in non-small cell lung cancer (NSCLC). Pathol Res Pract 2006;202:577–583. 38. Tsutsumida H, Goto M, Kitajima S, et al. MUC4 expression correlates with poor prognosis in small-sized lung adenocarcinoma. Lung Cancer 2007;55:195–203. 39. Majhi PD, Lakshmanan I, Ponnusamy MP, et al. Pathobiological implications of MUC4 in non-small-cell lung cancer. J Thorac Oncol 2013;8:398–407. 40. Damera G, Xia B, Ancha HR, Sachdev GP. IL-9 modulated MUC4 gene and glycoprotein expression in airway epithelial cells. Biosci Rep 2006;26:55–67. 41. Yamada N, NishidaY, Tsutsumida H, et al. Promoter CpG methylation in cancer cells contributes to the regulation of MUC4. Br J Cancer 2009;100:344–351. 42. Barsky SH, Cameron R, Osann KE, Tomita D, Holmes EC. Rising incidence of bronchioloalveolar lung carcinoma and its unique clinicopathologic features. Cancer 1994;73:1163–1170. 43. Kim S, Lewis C, Nadel JA. CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells. J Immunol 2011;186:3392–3400. 44. Kang JH, Lee EH, Park SW, Chung IY. MUC5AC expression through bidirectional communication of Notch and epidermal growth factor receptor pathways. J Immunol 2011;187:222–229. 45. Chen Y, Watson AM, Williamson CD, et al. Glucocorticoid receptor and histone deacetylase-2 mediate dexamethasone-induced repression of MUC5AC gene expression. Am J Respir Cell Mol Biol 2012;47:637–644. 46. Chen Y, Zhao YH, Di YP, Wu R. Characterization of human mucin 5B gene expression in airway epithelium and the genomic clone of the amino-terminal and 5’-flanking region. Am J Respir Cell Mol Biol 2001;25:542–553. 47. Iwashita J, Yamamoto T, Sasaki Y, Abe T. MUC5AC production is downregulated in NCI-H292 lung cancer cells cultured on type-IV collagen. Mol Cell Biochem 2010;337:65–75. 48. Bai CH, Song SY, Kim YD. The inhibitory effect of the leukotriene receptor antagonist on leukotriene D4-induced MUC2/5AC gene expression and mucin secretion in human airway epithelial cells. Auris Nasus Larynx 2007;34:203–206. 49. Skrzypek K, Tertil M, Golda S, et al. Interplay between heme oxygenase-1 and miR-378 affects non-small cell lung carcinoma growth, vascularization, and metastasis. Antioxid Redox Signal 2013;19:644–660. 50. Hamamoto A, Abe Y, Nishi M, et al. Aberrant expression of the gastric mucin MUC6 in human pulmonary adenocarcinoma xenografts. Int J Oncol 2005;26:891–896. 51. Rossi G, Murer B, Cavazza A, et al. Primary mucinous (so-called colloid) carcinomas of the lung: A clinicopathologic and immunohistochemical
Mucins in Lung Cancer
study with special reference to CDX-2 homeobox gene and MUC2 expression. Am J Surg Pathol 2004;28:442–452. 52. Fan H, Bobek LA. Regulation of human MUC7 mucin gene expression by cigarette smoke extract or cigarette smoke and Pseudomonas aeruginosa lipopolysaccharide in human airway epithelial cells and in MUC7 transgenic mice. Open Respir Med J 2010;4:63–70. 53. Situ D, Wang J, Ma Y, et al. Expression and prognostic relevance of MUC1 in stage IB non-small cell lung cancer. Med Oncol 2011;28:S596–S604. 54. Demirag F, Cakir E, Bayiz H, Eren Yazici U. MUC1 and bcl-2 expression in preinvasive lesions and adenosquamous carcinoma of the lung. Acta Chir Belg 2013;113:19–24. 55. Powell E, Chow LQ. BLP-25 liposomal vaccine: A promising potential therapy in non-small-cell lung cancer. Expert Rev Respir Med 2008;2:37–45. 56. Spira A, Beane J, Shah V, et al. Effects of cigarette smoke on the human airway epithelial cell transcriptome. Proc Natl Acad Sci USA 2004;101:10143–10148. 57. Khodarev NN, Pitroda SP, Beckett MA, et al. MUC1-induced transcriptional programs associated with tumorigenesis predict outcome in breast and lung cancer. Cancer Res 2009;69:2833–2837. 58. MacDermed DM, Khodarev NN, Pitroda SP, et al. MUC1-associated proliferation signature predicts outcomes in lung adenocarcinoma patients. BMC Med Genomics 2010;3:16. 59. Ohnishi H, Yokoyama A, Kondo K, et al. Comparative study of KL-6, surfactant protein-A, surfactant protein-D, and monocyte chemoattractant protein-1 as serum markers for interstitial lung diseases. Am J Respir Crit Care Med 2002;165:378–381. 60. Shields PG. Molecular epidemiology of lung cancer. Ann Oncol 1999;10:S7–11. 61. Matsumura N, Yamamoto M, Aruga A, Takasaki K, Nakano M. Correlation between expression of MUC1 core protein and outcome after surgery in massforming intrahepatic cholangiocarcinoma. Cancer 2002;94:1770–1776. 62. Weed DT, Gomez-Fernandez C, Bonfante E, et al. MUC4 (sialomucin complex) expression in salivary gland tumors and squamous cell carcinoma of the upper aerodigestive tract. Otolaryngol Head Neck Surg 2001;124:127–141. 63. Gao L, Liu J, Zhang B, et al. Functional MUC4 suppress epithelialmesenchymal transition in lung adenocarcinoma metastasis. Tumour Biol 2001;35:1335–1341.. 64. Hishida T, Ishii G, Kodama T, et al. Centrally located adenocarcinoma with endobronchial polypoid growth: Clinicopathological analysis of five cases. Pathol Int 2011;61:73–79. 65. Yu CJ, Shih JY, Lee YC, Shun CT, Yuan A, Yang PC. Sialyl Lewis antigens: Association with MUC5AC protein and correlation with post-operative recurrence of non-small cell lung cancer. Lung Cancer 2005;47:59–67. 66. López-Ferrer A, Curull V, Barranco C, et al. Mucins as differentiation markers in bronchial epithelium. Squamous cell carcinoma and adenocarcinoma display similar expression patterns. Am J Respir Cell Mol Biol 2001;24:22–29. 67. Szabo E. MUC1 expression in lung cancer. Methods Mol Med 2003;74:251–258. 68. Sangha R, Butts C. L-BLP25: A peptide vaccine strategy in non small cell lung cancer. Clin Cancer Res 2007;13:s4652–s4654. 69. Butts C, Murray N, Maksymiuk A, et al. Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol 2005;23:6674–6681. 70. Brossart P, Heinrich KS, Stuhler G, et al. Identification of HLA-A2restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies. Blood 1999;93:4309–4317. 71. Sharpe JC, Abel PD, Gilbertson JA, et al. Modulated expression of human leucocyte antigen class I and class II determinants in hyperplastic and malignant human prostatic epithelium. Br J Urol 1994;74:609–616. 72. Wu YL, Park K, Soo RA, et al. INSPIRE: A phase III study of the BLP25 liposome vaccine (L-BLP25) in Asian patients with unresectable stage III non-small cell lung cancer. BMC Cancer 2011;11:430. 73. Ramlau R, Quoix E, Rolski J, et al. A phase II study of Tg4010 (MvaMuc1-Il2) in association with chemotherapy in patients with stage III/IV non-small cell lung cancer. J Thorac Oncol 2008;3:735–744. 74. Kurosaki T, Higuchi N, Kawakami S, et al. Self-assemble gene delivery system for molecular targeting using nucleic acid aptamer. Gene 2012;491:205–209. 75. Rahn JJ, Dabbagh L, Pasdar M, Hugh JC. The importance of MUC1 cellular localization in patients with breast carcinoma: An immunohistologic study of 71 patients and review of the literature. Cancer 2001;91:1973–1982.
Copyright © 2014 by the International Association for the Study of Lung Cancer
27
