Mucin Production Correlates with Dual Expression of Epidermal Growth Factor Receptor and Its Ligand the Epidermal Growth Factor in Non-Small Cell Lung Cancer

Introduction: Mucoproduction plays an important role in the processes of tumor progression, invasion and metastasis. In non-small cell lung cancer (NSCLC), mucins hypersecretion has been associated with alterations in the epidermal growth factor receptor (EGFR) expression. However, the relation of mucins with EGFR in these tumors is not completely clarifi ed. Aims: To evaluate the relation between mucoproduction, the expression of EGFR and its ligand the epidermal growth factor (EGF) in NSCLC samples. Materials and Methods: A number of 71 routinely processed formalin-fi xed and paraffi n-embedded archival samples with diagnosis of NSCLC were used. Mucoproduction was considered when periodic acidSchiff reaction (PAS) positive and/or alcian blue positive substances were observed. The immunolocalization of EGFR and EGF molecules were detected by mean of immunohistochemical methods. Chi-square test was applied to compare two or three different parameters. In all cases, the criterion for statistical signifi cance was p < 0.05. Results: The production of mucins was related with the occurrence of metastasis (p = 0.0151) as well as, with a more advanced stage of NSCLC disease (p = 0.0409). The presence of mucin was statistically signifi cant associated to histology (p < 0.0001) and degree of cytological atypia (p = 0.0325). The expression of EGFR was associated with mucin hyperproduction (p = 0.0205). The levels of mucin production was statistical signifi cant increase in NSCLC samples displaying double expression of EGFR and EGF (p = 0.0297) when compared with EGFR-/EGF+ and EGFR-/EGF-. Conclusions: The results obtained in the present work could support the role of uncontrolled EGFR system activation mediated by the ligand EGF in NSCLC mucins hyperproduction as well as its relation with tumor progression and metastasis processes. Research Article Mucin Production Correlates with Dual Expression of Epidermal Growth Factor Receptor and Its Ligand the Epidermal Growth Factor in Non-Small Cell Lung Cancer Rancés Blanco1*, Charles E Rengifo2, Elizabeth Domínguez3, Damián Blanco4, Mercedes Cedeño1 and Enrique Rengifo1 1Laboratory of Recognition and Biological Activity Assays, Cuba 2Department of Pathology, Manuel Fajardo General Hospital, Zapata and D Street Vedado, Plaza de la Revolución, 10400 Havana, Cuba 3Laboratory of Biochemistry, Center of Molecular Immunology, 216 Street and 15 Avenue, Atabey, Playa, PO Box 16040, 11600 Havana, Cuba 4Department of Cell Biology and Tissues Banking, National Institute of Oncology and Radiobiology, 10400 Havana, Cuba Dates: Received: 18 February, 2017; Accepted: 19 April, 2017; Published: 20 April, 2017 *Corresponding author: Rancés Blanco Santana, PhD, Laboratory of Recognition and Biological Activity Assays, Center of Molecular Immunology, 216 Street and 15 Avenue, Atabey, Playa, PO Box 16040, Havana 11600, Cuba, Tel: (537) 2717933-3464; Fax: (537) 2720644; E-mail: rances@cim.sld.cu


Introduction
Lung cancer remains one of the leading causes of death from cancer worldwide [1]. There are two main variant of the disease, non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), but, NSCLC constitute about 80-85% cases of all lung carcinomas [2,3]. Most  Mucins constitute a heterogeneous group of highly O-glycosylated macromolecules synthesized by epithelial cells [5]. Airway mucins are major components of the soluble layer and/or viscoelastic gel that comprise lung mucus in healthy airways and contribute to the mucociliary defense system that protects the lungs against pathogens and environmental toxins [6]. Particularly, increased hyperproduction of secretory mucins usually occurs in lung tumors [5,7]. Nevertheless, their cellular localization and variations along the respiratory tract or occurring in respiratory diseases have received little attention.
Mucoproduction plays an important role in the processes of tumor progression, invasion and metastasis. In addition, mucins are engaged in malignant cells survival and protection against the host immune response [8], contributing to the aggressive potential of tumors. Airway obstruction from mucus accumulation is one of the major pathological changes in NSCLC [9]. In line with this, chronic mucus hypersecretion has been considered a signifi cant predictor of death from these tumors [10]. However, further investigations in order to elucidate the biological signifi cance of mucins in carcinomas have been suggested [11].
Previously, the epidermal growth factor receptor (EGFR) mutation was considered a critical event in the pathogenesis of nonmucinous bronchioloalveolar carcinoma (BAC) [12] while, invasive mucinous pattern and extracellular mucin have been associated with KRAS mutation in lung adenocarcinoma [13]. Moreover, the lepidic-predominant group was associated with EGFR mutation compared with nonlepidic-predominant tumors [13]. Furthermore, one preliminary study demonstrated a signifi cant correlation between nonmucinous BAC histology and EGFR expression [14]. Nevertheless, the relation of mucins with EGFR expression in lung cancer is not complete clarifi ed [7].
In this study, it was evaluated the relation between mucoproduction and the expression of EGFR in NSCLC patients. Additionally, it was assessed the role of the double expression of EGFR and its ligand the EGF in mucins hyperproduction.

Materials and Methods
Tissue specimens. A number of 71 routinely processed formalin-fi xed and paraffi n-embedded archival samples with diagnosis of NSCLC were received from the pathology department of both Hermanos Ameijeiras General Hospital and the National Institute of Oncology and Radiobiology, after approved consent by the institutional ethical committees. Five micrometer serial sections from each block were obtained in a micrometer (Leitz 1512) and they were mounted on plus slides (Dako S2024). All sections were attached to the slide by heating in a 60ºC oven for 1h. Afterwards, the slides were dewaxed in xylene and rehydrated in graded ethanol series in the usual way. The samples were maintained in tap water until they were stained.
Pathological features. The evaluation of some pathological features was performed for each tumor tissues using the hematoxylin and eosin (HE) staining. Morphologic parameters such as histopathological classifi cation and degree of histologic differentiation were evaluated. In addition, some cytomorphologic characteristics such as cell and nuclear size, cellular shape, chromatin pattern, nucleoli, and amount of cytoplasm were assessed. This measured was considered as the degree of cytological atypia and it was subjectively expressed as low, moderate and high. Immunohistochemical staining. The immunolocalization of the epidermal growth factor receptor (EGFR) and its ligand the epidermal growth factor (EGF) was performed as it was previously described in [15]. Briefl y, the slides were pre-treated with 0.4% pepsin in 0.1N hydrochloric acid solution at 37°C for 30 minutes. Afterward, the tissues were incubated with ior egf/ r3 Mab (anti-EGFR) and CB-EGF1 (anti-EGF ligand) in a humid chamber for 1h at room temperature followed by a rabbit anti- were grouped as follow: low expression (scores < 150) and high expression (scores ≥ 150) as it was previously described in [16] (

Results
Patients characteristics. Table 1 Table   3. Consequently, the expression of EGFR was associated with mucin hyperproduction (p = 0.0205, Chi-Square test) ( Figure   2).   Table 3. Consequently, the levels of mucin production was statistical signifi cant increased in NSCLC samples displaying double expression of EGFR and its ligand the EGF (p = 0.0297, Chi-Square test) (Figure 3).

Discussion
Mucins have been implicated in the pathogenesis of epithelial cell malignancies [17]. Alterations of the expression pattern of mucins have been described in carcinomas as well as in their precursor lesions [18,19]. Airway obstruction from mucus accumulation or from a tumor projecting into a bronchus is one of the major pathological changes in lung cancer [9]. Specifi cally, chronic mucus hypersecretion has been considered a signifi cant predictor of death from lung cancer [10]. Mucinous carcinoma has been known to have a propensity for higher incidence of lymph node metastasis, venous and lymphatic invasions, local recurrence and distant metastasis compared with nonmucinous tumors [20].
The detection of certain type of mucins (MUC) by means of immunohistochemical methods has been reported to be associated with the progression of lung adenocarcinoma [21]. In a previous work, our group demonstrated that PASpositive substances correlated with the appearance of distant metastasis in NSCLC patients [22]. Here, the occurrence of mucin production was related with the appearance of distant metastasis but also with a more advanced disease. In line with this, Yu et al., reported that MUC5B and MUC5AC overexpressing tumors tended to increase the risk of distant metastasis and decrease overall survival from lung cancer, compared with nonexpressing tumors [23]. Moreover, aberrant MUC1 expression was associated with disease progression [24] and with a poor prognosis [25,26] while, up-regulation of MUC6 expression was related with tumor size in NSCLC patients [21].
In the present study, the mucin production was mostly evidenced in adenocarcinomas as compared with the rest of lung tumors, similar to previous reports [22,27,28]. Interestingly, the presence of mucin was signifi cantly higher in NSCLC samples displaying increased degree of cytological atypia.
Previously, it was published that transmembrane mucins (e.g MUC1 and MUC4), can signal the disruption of tight junctions and adherens junctions affecting polarity as well as both cellcell and cell-extracellular matrix interactions [19,29]. It have been also reported that overexpression of MUC1 in cancer cells alter the function of -catenin/E-cadherin pathway in adherens junctions [30] inducing actin cytoskeleton alterations [31]. In this sense, the loss of polarity and cell-cell interactions could alter the cell morphology allowing to an increased cell pleomorfi sm and also favors the development of metastasis [32].
EGFR cascade is involved in mucin production as well as in the repair of damaged airway epithelium by a wide variety of stimuli [33]. Changes that occur during lung carcinogenesis usually conduce to dys-regulation of both mucin and EGFR expressions [5,34]. In this study, the expression of EGFR was     [35]. However, in previous studies the EGFR mutation was related with nonmucinous bronchioloalveolar carcinoma [12,14] as well as with the lepidic-predominant form of this tumor [13].
On the other hand, the contribution of EGFR and its ligands in mucin production in normal airway epithelium using AB-PAS reaction was previously reported [36]. Co-expression of EGFR and its ligands results in activation of an autocrine system leading to dysregulated EGFR action and uncontrolled both tumor growth [37] and secretion [38]. It is known that mucins expression (e.g. MUC2 and MUC5AC) is controlled by EGF family and mitogen-activated protein kinase (MAPK) cascade [38]. Moreover, it has been reported that upregulated expression of mucins, such as MUC2 and MUC5AC, bind with EGFR and activate the Ras/Raf pathway [38,39]. In line with this, our group previously reported that the dual expression of EGFR and its ligand the EGF is related with increased cell proliferation and with a more aggressive behavior of NSCLC [16].
Interestingly, using selective inhibitors of EGFR tyrosine kinase phosphorylation, it was reported that EGFR ligand induced mucin MUC5AC synthesis is dependent on EGFR activation [36]. In this work, NSCLC samples displaying the phenotype EGFR + /EGF + were associated with a more accentuated production of mucins as compared with EGFR + /EGFand EGFR -/ EGFtumors. In line with this, EGFR phosphorylation upon binding to EGF and TGF- resulted in a remarkable increase of MUC2 and MUC5AC mRNAs levels, promoter activity, and apomucin expression in the mucoepidermoid NCI-H292 lung cancer cell line [38]. In this sense, our results could support the role of uncontrolled EGFR system activation mediated by the ligand EGF in NSCLC mucins hyperproduction.
In view of that, it was suggested that disrupting the EGFR cascade that leads to mucus production is benefi cial in airway hypersecretory diseases [40]. Interestingly, there are two different immunotherapeutic approaches registered in Cuba targeting the EGFR/EGF system. The fi rst one is a molecular vaccine that induces anti-EGF antibodies neutralizing endogenous EGF (CIMAVax-EGF) [41]. The other one is a humanized monoclonal antibody that directly binds the extracellular domain of the EGFR (nimotuzumab) [42]. Interestingly, promising results in advanced NSCLC patients treated with CIMAVax-EGF [43,44] or nimotuzumab [45,46] has been obtained. In this way, the evaluation of mucin production in NSCLC patients after CIMAVax-EGF or nimotuzumab treatment seems to be of utmost importance.

Conclusions
The association between mucin hyperproduction and a more advanced state of NSCLC disease was demonstrated.
Interestingly, mucoproduction was also related with the EGFR as well as with the double expression of EGFR and its ligand the