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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0b013e3182829e06
Original Articles

Pathobiological Implications of MUC4 in Non–Small-Cell Lung Cancer

Majhi, Prabin Dhangada*; Lakshmanan, Imayavaramban*; Ponnusamy, Moorthy P.*; Jain, Maneesh*; Das, Srustidhar*; Kaur, Sukhwinder*; Shimizu, Su Tomohiro*; West, William W.; Johansson, Sonny L.†,‡; Smith, Lynette M.§; Yu, Fang; Rolle, Cleo E.; Sharma, Poonam#; Carey, George B.; Batra, Surinder K.*,‡; Ganti, Apar Kishor**††

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Author Information

*Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE; Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE; §Center for Collaboration on Research, Design and Analysis, College of Public Health, University of Nebraska Medical Center, Omaha, NE; Department of Biostatistics College of Public Health, University of Nebraska Medical Center, Omaha, NE; Section of Hematology-Oncology Department of Medicine, University of Chicago, Chicago, IL; #Department of Pathology and Creighton Medical Laboratories, Creighton University Medical Center, Omaha, NE; **Department of Internal Medicine, VA Nebraska-Western Iowa Health Care System, Omaha, NE; ††Division of Oncology-Hematology, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE.

Prabin Dhangada Majhi and Imayavaramban Lakshmanan contributed equally to this article

Disclosure: This work is supported by a grant from the US Department of Veteran Affairs.

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 68198-7680. E-mail: aganti@unmc.edu

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Abstract

Introduction: Altered expression of MUC4 plays an oncogenic role in various cancers, including pancreatic, ovarian, and breast. This study evaluates the expression and role of MUC4 in non–small-cell lung cancer (NSCLC).

Methods: We used a paired system of MUC4-expressing (H292) and MUC4-nonexpressing (A549) NSCLC cell lines to analyze MUC4-dependent changes in growth rate, migration, and invasion using these sublines. We also evaluated the alterations of several tumor suppressor, proliferation, and metastasis markers with altered MUC4 expression. Furthermore, the association of MUC4 expression (by immunohistochemistry) in lung cancer samples with patient survival was evaluated.

Results: MUC4-expressing lung cancer cells demonstrated a less proliferative and metastatic phenotype. Up-regulation of p53 in MUC4-expressing lung cancer cells led to the accumulation of cells at the G2/M phase of cell cycle progression. MUC4 expression attenuated Akt activation and decreased the expression of Cyclins D1 and E, but increased the expression of p21 and p27. MUC4 expression abrogated cancer cell migration and invasion by altering N- & E-cadherin expression and FAK phosphorylation. A decrease in MUC4 expression was observed with increasing tumor stage (mean composite score: stage I, 2.4; stage II, 1.8; stage III, 1.4; and metastatic, 1.2; p = 0.0093). Maximal MUC4 expression was associated with a better overall survival (p = 0.042).

Conclusion: MUC4 plays a tumor-suppressor role in NSCLC by altering p53 expression in NSCLC. Decrease in MUC4 expression in advanced tumor stages also seems to confirm the novel protective function of MUC4 in NSCLC.

MUC4 (mucin 4) is a high–molecular-weight type-I transmembrane protein. The canonical MUC4, i.e., the longest isoform, is expressed as a single propeptide that is predicted to undergo cleavage and dimerization. The resulting mucin-like subunit MUC4α with an N-terminal domain, unique 16-mer tandem repeat (TR) domain of variable length, a nidogen-like (NIDO) domain, and an adhesion-associated domain (AMOP) dimerizes with the growth factor–like subunit MUC4α carrying von-Willebrand factor and three epidermal growth factor and epidermal growth factor–like domains and anchors to the cell membrane with a 21-mer transmembrane domain of MUC4β.1 In addition, MUC4β carries a short (22-mer) cytoplasmic tail. The MUC4 TR domain is rich in proline, threonine, and serine residues and is a target for O-linked glycosylation, which could amount to 50% of the total molecular weight of MUC4.

MUC4 is expressed by the epithelia of the tubular organs such as lung,2 colon,3 cervix,4 cornea,5 prostate,6 middle ear, and Eustachian tube.7 However, the functional role of MUC4 has been best studied in malignancies of organs that normally lack MUC4 expression including pancreas,8 ovary,9 and breast.10 In pancreatic cancer, a novel gain of function due to aberrant overexpression and/or loss of cell polarity enabling MUC4 to physically interact with, activate (by phosphorylation), and stabilize HER2, a ligand-dependent receptor tyrosine kinase, seems to be the principal mechanism of MUC4-associated pathogenesis.1,11

MUC4 expression is observed at all levels in the respiratory tract, i.e., trachea, main bronchus, lobar bronchus, smaller bronchi, and bronchioles, but is undetectable in submucosal glands and alveolar epithelial cells.12 MUC4 is highly expressed in the surface epithelium lining, i.e., in ciliated cells, goblet cells, and basal cells.13 MUC4 expression was found to be associated with precancerous lesions like squamous cell metaplasia, dysplasia, and also with well-differentiated squamous cell carcinoma.14 However, MUC4 expression in neoplastic lung seems to vary based on histologic subtypes. Among the non–small-cell lung carcinomas (NSCLC), MUC4 expression was more characteristic of adenocarcinoma than squamous cell carcinoma.15 Clinically, MUC4 expression in small lung tumors was found to correlate with shorter disease-free survival and poor survival.16 However, in another study, an increase in MUC4 immunoreactivity was found to be associated with a longer patient survival.15 Given these discrepancies in the literature, we have investigated the role and mechanism of MUC4 in lung cancer progression.

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PATIENTS AND METHODS

The study was approved by the Institutional Review Board and other regulatory committees at the VA Nebraska-Western Iowa Health Care System.

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Cell Culture and Transfection

Human NSCLC cell lines NCI-H292 and A549 were purchased from the American Type Culture Collection (Manassas, VA). Stable clones of NCI-H292 in which MUC4 was down-regulated using small-hairpin RNA against target sequence (5′-CAGCGACACTAGAGGGACA-3′) located 151 bp downstream of ATG (H292-ShMUC4) and scramble transfected control sublines expressing endogenous MUC4 (H292-Scr) were established as described previously.17 Stable clones of mini-MUC4 transfected A549 (A549-MUC4) overexpressing an estimated 320 kDa MUC4 protein containing all domains of wild-type MUC4 but only 10% of the TRs and empty vector control (A549-PsecTagC) were established as described earlier.18 NCI-H292 cells were routinely grown in RPMI-1649 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% complement-inactivated fetal bovine serum (FBS), 100 units of penicillin, and 100 µg/ml streptomycin. A549 cells were grown in F12K media supplemented with 15% FBS, 100 units of penicillin, and 100 µg/ml of streptomycin. Media of transfected cell lines were supplemented with either 1 µg/ml puromycin (NCI-H292) or 200 µl/ml zeocine (A549).

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Proliferation Assay

For growth curve analyses, cells were seeded in six-well plates at a density of 1 × 103 cells/well. After 24 hours of growth, media were changed to 1% FBS in RPMI (H292-Scr and H292-ShMUC4) and 5% FBS in F12K (A549-Vector and A549-MUC4). Viable cell numbers from triplicate wells were determined every 24 hours for 6 days by trypan blue exclusion using a Vi-Cell XR instrument (Beckman Coulter, Fullerton, CA). For cell cycle analyses, 1 × 106 cells were grown briefly in 10-mm plates and synchronized with 2 mM thymidine for 12 hours, followed by treatment with 2-deoxycytidine 9 hours and a second 2 mM thymidine block for 14 hours. Cells were released in complete media for 48 hours, collected, fixed in 70% ethanol, and stained with Telford reagent (90 mM ethylenediaminetetraacetic acid, 2.5 mU of RNase A/ml, 50 mg of propidium iodide/ml, and 0.1% Triton X-100 in phosphate-buffered saline [PBS]). DNA content was then analyzed by fluorescence-activated cell sorting analyses.

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Cell Motility Assay

In serum-free media, 1 × 106 cells/chamber were plated on the noncoated polyethelene terapthalate (PET) membrane chambers (8 µm, Becton Dickinson, Franklin Lakes, NJ), inserted into six-well plates containing 10% FBS in media, and grown for 24 hours. The PET inserts were washed and stained in Diff-Quick cell staining kit (Dade Berhing Inc., Newark, DE) and cells that did not migrate were scrapped off with cotton swabs. Cells that traversed and stained were counted in 10 random fields of view at 10× magnification, in a phase-contract microscope and expressed as number of cells per view. For the in vitro scratch assay, H292-Scr and H292-ShMUC4 cells were grown at 6 × 106 in 10-cm cell plates. Cells were imaged at 0, 24, and 48 hours of growth. H292-Scr cells showed less migration than H292-ShMUC4 cells at corresponding time points.

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Cell Invasion Assay

The invasive potential of the NSCLC cells was estimated by their ability to penetrate PET membrane coated with a thin layer of Matrigel (BD Biocoat Matrigel six-well cell invasion chamber). For each experiment, 1 × 105 cells/well in 2 ml of RPMI media (or F12K media) containing serum-free media were loaded onto the top chamber and 2 ml of complete RPMI media (F12K media) was added in the bottom chamber. After 24 hours of incubation, invasive cells reaching the lower side of the PET membrane were stained and measured as described earlier.

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Quantitative Real-Time Polymerase Chain Reaction Analyses

Total cellular RNA was isolated from cells grown in complete RPMI media for 48 hours using RNeasy kit (Qiagen, Valencia, CA), according to the manufacturer’s instruction. Total RNA was reverse-transcribed and quantitative real-time polymerase chain reaction was performed using SYBR Green method in a LightCycler 480 II (Roche, Basel, Switzerland).

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Apoptosis Assay

Apoptosis was measured by Annexin-V-FLUOS staining kit (Roche diagnostics, Indianapolis, IN). Cells (1 × 106/10-cm plate) were incubated overnight and grown in 1% serum for the next 48 hours. Cells were collected and stained with Annexin-V and propidium iodide solution, and the percentage of apoptotic and necrotic cells was measured by fluorescence-activated cell sorting analyses.

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Immunoblot Analyses

Cells were typically grown for 48 hours in complete media, unless mentioned otherwise, and total protein lysate was collected in RIPA lysis buffer (50 mM Tris–Cl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.25% Na-deoxycholate; 1 mM ethylenediaminetetraacetic acid; 1 mM phenylmethylsulfonyl fluoride; 1 µg/ml aprotinin and leupeptin; 1 mM Na3VO4; and 1 mM NaF) and passed through a 25-gauge syringe several times. Total protein content was assayed with the Bio-Rad DC Protein assay (Bio-Rad, Hercules, CA). Protein lysates (40 µg/ml) were loaded onto a sodium dodecyl sulfate–agarose gel (2%) under reduced conditions to resolve and immunoblot for MUC4. All other proteins were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%). Resolved proteins were transferred to polyvinylidene difluoride membrane and blocked in nonfat milk (5%) for 1 hour. The membrane was incubated in the appropriate primary antibody according to the manufacturer’s instructions for MUC4 at 1 µg/ml in PBS for 2 hours at room temperature, followed by 4 × 10 minute washes in Tris-buffered saline Tween-20 (50 mM Tris–Cl, pH 7.4; 150 mM NaCl, and 0.05% Tween-20). The membrane was incubated for 1 hour with the appropriate secondary antibody conjugated with horseradish peroxides at the dilution of 1:2000 in PBS, followed by 4 × 10 minute washes in Tris-buffered saline Tween-20. Enhanced chemiluminiscence reagents (Amersham Biosciences, Piscataway, NJ) were applied on the membrane as per the manufacturer’s instructions, and the blot was exposed to an ECL-sensitive film (Biomax Films, Kodak, Rochester, NY).

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Tissue Microarrays and Immunohistochemistry

A lung cancer tissue microarray (TMA) was obtained from US Biomax, Inc. (Rockville, MD) The TMA (Cat. No. BC0402) has a total of 100 tissues from a spectrum of malignant, nonmalignant lung diseases, and normal lung, including 20 cases each of squamous cell carcinoma and adenocarcinoma of varying grades, 10 cases each of small cell undifferentiated carcinoma, alveolar cell carcinoma, and metastatic squamous cell carcinoma, and 5 cases each of carcinoid, inflammatory pseudotumor, tuberculosis, cancer adjacent tissue, cancer adjacent normal tissue, and normal lung tissue. In addition, samples were obtained from patients with stages I–III NSCLC from the University of Chicago specimen bank (Courtesy: Dr. Ravi Salgia). MUC4 expression was correlated with tumor stage and survival. In addition, where paired samples were available from the tumor and corresponding lymph node, MUC4 expression between the two was compared.

As per recommendations of the manufacturer, before immunohistochemistry, the slides were baked at 60°C for 2 hours. Then, slides were deparaffinized using EzDewax (Bio Genex, Fremont, CA) for 30 minutes. Sections were hydrated through graded alcohol and endogenous peroxidase activity was quenched by incubating the sections in 0.3% H2O2 in methanol for 30 minutes. After washing the slides in PBS (5 minutes × 2), antigen was retrieved by heating the slides in citrate buffer (0.01 M, pH 6.0) at 80°C for 20 minutes. After heating, the samples were allowed to cool for 15–20 minutes in room temperature, followed by washing with PBS (5 minutes × 2). Nonspecific binding was blocked by incubating the sections with 2.5% horse serum for 30 minutes (Impress reagent Kit, Vector, CA). This was followed by washing with PBS (5 minutes × 2). Sections were then incubated with anti-MUC4 monoclonal antibody (1:500) at 4°C overnight. The slides were washed and incubated with secondary antibody (peroxidase-labeled Universal antimouse/antirabbit IgG) (Vector, Burlingame, CA) for 30 minutes. Sections were then washed with PBS followed by treatment with diamino benzidine reagents (0.2 mg/ml) and incubated for 10 minutes. After washing with distilled water, counterstaining was done by using hematoxylin (Vector). After washing in tap water, sections were dehydrated in graded alcohol, and after air drying, the slides were mounted in Permount permanent mounting media (Fisher Scientific, Fair Lawn, NJ). All slides were observed under Nikon E400 light microscope and representative photographs were taken.

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Statistical Analyses

All experiments were performed in triplicates, and the statistical significance of the results was analyzed using Student’s t test or Mann–Whitney test. For patient samples, Mann–Whitney test was used to compare composite score between groups. For paired comparison of composite score between metastatic and primary tumor, the Wilcoxon signed rank test was used. Chi-square tests were conducted for categorical data. Overall survival is defined as time from diagnosis to death or last follow-up. The Kaplan–Meier method was used to estimate overall survival distributions and the log-rank test was used to compare survival distributions between groups. Pair-wise p values were adjusted for multiple comparisons with Bonferroni’s method. A p value of less than 0.05 was considered to be statistically significant.

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RESULTS

MUC4 Expression in Lung Cancer

To investigate the causal relationship of MUC4 down-regulation with tumor progression in vitro, a panel of eight NSCLC lung cell lines was screened for MUC4 expression, of which H292 and HCC827 cell lines showed high level of expression by quantitative real-time polymerase chain reaction (Fig. 1A). MUC4 expression was silenced in H292 by retroviral RNAi method as described previously.1 Stable ectopic expression of the MUC4 gene containing all unique domains of MUC4 along with 10% of the TR was obtained by transfection in a MUC4-nonexpressing cell line A549. Knockdown of endogenously expressed MUC4 in H292 and the ectopic expression of MUC4 in A549 were confirmed by Western blot and confocal analysis (Fig. 1B).

Figure 1
Figure 1
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MUC4 Attenuates In Vitro Proliferation of NSCLC Cells

Growth rates of H292-ShMUC4 and A549-MUC4 cells were compared with those of control H292-Scr and A549-Vector, respectively. MUC4 knockdown H292 cells showed a significantly increased cell growth rate compared with control cells (p = 0.011) (Fig. 2A). Similarly, MUC4-expressing A549-MUC4 cells showed significantly decreased cell growth compared with control A549-Vector cells (p = 0.053) (Fig. 2B). Decreased growth rates in the presence of MUC4 could result either from an impairment of cell cycle progression or an increase in cell death. Cell cycle analyses after synchronization with double-thymidine block showed a higher percentage of cells in S-phase in H292-ShMUC4 cells (Fig. 2C) as compared with H292-Scr cells (p = 0.034). Similarly, A549-PsecTagC cells showed increased S-phase population (Fig. 2D) compared with A549-MUC4. These results suggest that expression of MUC4 suppresses lung cancer cell growth.

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MUC4 Abrogates Proliferative Signals in Lung Cancer

Previous studies on MUC4 in different cancers have shown that MUC4 interacts with and stabilizes HER2, which mediates downstream proliferative signals such as activation of PI3K/Akt and MEK/Erk pathway.1 Because our studies imply an opposite function of MUC4 in NSCLC, we hypothesized that MUC4 plays a tumor-suppressor role in lung cancer. In contrast to the previous study, pY1248HER2 and total HER2 levels were unaffected (Fig. 3A). However, with MUC4 down-regulation, a significant increase in phosphorylation of Akt (Ser473) was observed while total Akt level was unchanged (Fig. 3B). Activated Akt phosphorylates multiple downstream targets to stimulate cell proliferation. These functions include phosphorylation and proteasomal targeting of p21Cip1 and p27Kip1, and inactivation of GSK3β that stabilizes G1 cyclins (Cyclin D and E) and transcription factors c-Jun and c-Myc.19 We observed elevated levels of inactive pSer9-GSK3β and Cyclin D and A, and a decrease in p21Cip1 and p27Kip1 in both MUC4 knockdown (H292-ShMUC4) and MUC4-nonexpressing (A549-Vector) cells, compared with their MUC4-expressing sublines (Fig. 3B).

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In a previous study on the role of MUC4 in pancreatic cancer cell line CD18/HPAF, transcriptome analyses showed 2.5-fold increases in TP53 after down-regulation of MUC4.17 Results of our study show that p53 mRNA and protein were more drastically decreased in MUC4 knockdown H292 cells than in control cells, and conversely, MUC4 expression increased p53 level (Fig. 3B and C), suggesting that MUC4 may transcriptionally regulate p53 expression.17 In addition, as a result of HER2/neu overexpression, Akt interacts and phosphorylates MDM2 and increases p53 degradation,20 suggesting an opposite function of MUC4 in lung cancer compared with pancreatic cancer. Moreover, based on this finding, we suggest that the interplay of MUC4 with p53 mediates the tumor-suppressor role of MUC4 in lung cancer. Expression of MUC4 attenuated the activation of Cyclins D1 and A, resulting in the decreased cancer cell proliferation compared with cells lacking MUC4 (Fig. 3B).

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NSCLC Cell Migration and Invasion Properties are MUC4 Dependent

To determine if MUC4 affects cell migration, we performed a wound-healing assay. A scratch was made in H292-ShMUC4 or H292-Scr cells grown to 90% confluency on a 10-mm plate, and the migration of cells to the scratch wound was observed over time. We observed that H292-ShMUC4 cells covered the scratch faster than the control (Fig. 4A). To corroborate this result, quantitative cellular motility assay was performed using Boyden chamber with PET membrane. H292-ShMUC4 and A549-Vector cells showed increased motility compared with H292-Scr (p = 0.0047) and A549-MUC4 (p = 0.0036), respectively (Fig. 4B). Invasive properties of both NSCLC cell lines were estimated with in vitro invasion assay. H292-ShMUC4 and A549-Vector cells showed more cells traversing the Matrigel-coated PET membrane than cells expressing MUC4 (Fig. 4C).

Figure 4
Figure 4
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To determine the cause of altered motility and invasive potential of MUC4-expressing lung cancer cells at the molecular level, confocal and Western blot analyses for markers of epithelial (E-cadherin/CDH1 and CK-18) and mesenchymal (N-cadherin/CDH2) phenotype were performed. The results show that the distribution of the epithelial marker E-cadherin was drastically decreased, whereas mesenchymal marker N-cadherin expression was high in MUC4 knockdown cells (Fig. 5A, left panel). Similar results were observed in MUC4-overexpressing A549 and A549-PsecTagC cells with the epithelial marker CK-18 and N-cadherin (Fig. 5A, right panel). Western blot results confirmed these findings (Fig. 5B). These results strongly suggest that MUC4-expressing lung cancer cells have less-invasive and motile properties because of the alteration of these markers.

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Binding of β1 integrin to collagen ligand activates intracellular signaling such as phosphorylation of FAK and Src.21 Phosphorylation of FAK at Y925 and Y577 was high in MUC4 knockdown cells (high invasive and motile property) compared with control cells (Fig. 5C), suggesting that in the absence of MUC4, pFAK is activated and potentially mediates the metastasis of lung cancer. The total FAK, however, was unchanged (Fig. 5C).

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MUC4 Expression Gradually Decreases with Increasing Stage of Malignancy

Owing to the contradictions in MUC4 expression and its correlation with tumor progression in the previous studies, we evaluated MUC4 expression in a commercially available lung cancer tissue array by immunohistochemistry. A total of 100 tissue spots were examined comprising the cancer stages I (n = 29), II (n = 15), III (n = 21), and IV (n = 10) and nonmalignant lung tissues (n = 25). We detected least MUC4 immunoreactivity in the nonmalignant specimens (mean composite score: 0.08), whereas MUC4 immunoreactivity was found in all stages of cancer (Fig. 6). A gradual decrease in the expression was observed with the progression of disease (mean composite score: stage I, 2.4; stage II, 1.8; stage III, 1.4; and metastatic, 1.2; p = 0.0093).

Figure 6
Figure 6
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MUC4 Expression Is Associated with Improved Survival

An additional 167 samples from 54 nonmetastatic patients with NSCLC with available clinical data were analyzed for MUC4 expression. Of these, 13 patients (25%) had stage I, 33 patients (63%) had stage II, and six patients (12%) had stage III disease. Distribution of the composite score differed significantly by stage (p = 0.0043). A higher proportion of patients with stage I NSCLC had the maximum possible score of 12 (77%) compared with stage II (18%) and stage III (57%) (p = 0.0001). On pair-wise comparison, patients with stage I NSCLC had a significantly higher proportion of composite scores of 12 as compared with stage II (p = 0.0009), but not stage III (p = 1.0). The proportion of patients with a composite score of 12 were not significantly different between patients with stage II and III (p = 0.16). In the 26 patients who had data for both the primary tumor and corresponding metastatic lymph node, MUC4 expression was higher in the primary tumor compared with the metastasis (p = 0.05). The effect of MUC4 expression on survival was analyzed in 29 patients with survival data. Of these, 16 had a composite score of 12, whereas 13 had a score less than 12. Patients with a score less than 12 had significantly worse survival than those with a score of 12 (p = 0.042) (Fig. 7).

Figure 7
Figure 7
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DISCUSSION

The objective of this study was to investigate the role of MUC4 on lung cancer cell proliferation and metastatic phenotype. The results demonstrate that MUC4 expression inhibits lung cancer cell proliferation and metastatic behavior in two separate lung cancer cell lines. These results seem to be confirmed by data from clinical specimens. Although H292 is an epithelial-type primary cancer cell line, A549 is a mesenchymal-type cell line.22 MUC4 down-regulation seems to transform epithelial to mesenchymal phenotype, whereas MUC4 expression reverses the phenotype in A549-MUC4 cells. To the best of our knowledge, this is the first investigation of the role of MUC4 as a tumor suppressor in NSCLC.

MUC4 has been implicated for oncogenic function in many cancers including pancreas,1 ovarian,23 and breast.24 In this study, we have seen that expression of MUC4 inhibits lung cancer cell proliferation by down-regulating the cell cycle proteins such as Cyclin A and Cyclin D1. Cyclin-dependent kinase inhibitors p21 and p27 were strongly up-regulated in MUC4-expressing lung cancer cells, suggesting that MUC4 plays a tumor-suppressor role in lung cancer by altering cell cycle regulator proteins. The decreased expression of GSK3β and phosphorylation of Akt in MUC4-expressing lung cancer cells suggests that expression of MUC4 suppresses lung cancer cell proliferation through regulation of GSK3β and Akt activation. A previous study demonstrated that Akt activation regulates cancer cell proliferation by proteasomal targeting of p21Cip1 and p27Kip1 and that loss of GSK3β stabilizes G1-phase cell cycle proteins (Cyclin D and E) and transcription factors c-Jun and c-Myc during cancer cell progression.19

Tumor-suppressor p53 is mutated in 50% of patients with lung cancer,25 which suggests that in a large subsection of lung cancer cases, the function of p53 is compromised. Recently, high levels of wild-type P53 expression in melanoma were shown to be transcriptionally inactive.26 Our results suggest that MUC4 induces p53, resulting in G2/M arrest as evident from the higher percent of G2/M cells in MUC4-expressing lung cancer cells. Data from this study suggest that MUC4 regulates p53 function as part of its tumor-suppressor role. Similarly, we have seen a decreased growth rate in MUC4-expressing lung cancer cells. The positive regulation of p53 expression and decreased growth rate of lung cancer cells by MUC4 could be of therapeutic importance in the future.

On studying the effect of MUC4 expression on the metastatic potential of lung cancer cells, we found that the most significant effect of MUC4 was on cell migration and invasion, with a significant decrease in MUC4-expressing cells and a corresponding increase in MUC4-nonexpressing lung cancer cells. These results clearly suggest that expression of MUC4 significantly inhibits the invasive and motile properties of lung cancer cells. Furthermore, evaluation of the epithelial and mesenchymal marker profile indicates that N-cadherin expression was up-regulated in MUC4 knockdown H292 cells along with a corresponding decrease in expression of E-cadherin. In MUC4-overexpressing A549 cells, the epithelial marker CK-18 expression was increased and N-cadherin was decreased compared with MUC4-nonexpressing control cells. These results suggest that expression of MUC4 suppresses N-cadherin expression, thereby resulting in decreased invasive and motile properties of lung cancer cells. Furthermore, activated FAK is increased in MUC4 knockdown cells, which showed highly invasive and motile properties. These results suggest that increased motility of lung cancer cells may be due to the activation of FAK-signaling pathway in the absence of MUC4.

In epithelial malignancies, altered mucin expression is significantly correlated with tumor progression. In tissues lacking MUC4 expression such as pancreas, breast, and ovary, MUC4 expression is observed after the onset of cancer and correlates with cancer progression and confers a poor prognosis.23,24,27 In contrast, MUC4 is the first mucin to be expressed in the lung, as early as 6.5 weeks of gestation in the embryonic foregut before organogenesis and cytodifferentiation in the primitive lung bud.12 MUC4 expression is retained to adulthood, when it is expressed strongly in the upper airways and moderately in bronchioles.

In our studies with the commercial TMA, we found a significant decrease in MUC4 expression with advanced stages of the disease with metastatic lesions expressing the least MUC4. However, we could not evaluate MUC4 expression at the inception of tumorigenesis, as our normal tissue specimens comprised mainly of lower bronchioles and terminal air sacs where MUC4 is virtually absent. Of note, the cancer adjacent normal tissue also lacked MUC4 expression.

Similarly, staining patterns from the patient samples show that a significantly higher proportion of patients with stage I disease had a composite score of 12 (maximum intensity and extent of staining). Moreover, patients with a composite score of 12 had a better survival as compared with those with a score of less than 12. Unfortunately, due to an inadequate number of samples, we were unable to adjust for stage, but the results seem to suggest that at the very least, MUC4 does not adversely affect lung cancer outcomes. This is also borne out by our finding that in patients with paired samples, the primary tumor had a greater composite MUC4 score than the corresponding metastatic lymph node. This would suggest loss of MUC4 expression as the lung cancer cells metastasize out of their primary site. Another hypothesis would be that the presence of MUC4 prevents spread of the tumor cells outside the primary.

Previous studies lack a consensus on the significance of MUC4 expression on lung cancer progression. Elevated levels of MUC4 mRNA were associated with poor prognosis.16 On the contrary, other studies have shown that MUC4 expression was associated with better survival and that reduced expression was found in all non–small-cell carcinomas.28 It is hoped that our results based both on in vitro studies and patient samples will put to rest the controversy regarding the role of MUC4 in NSCLC.

In conclusion, we found a significant increase of metastatic/proliferative stimuli in the absence of MUC4 expression, suggesting a tumor-suppressor role of MUC4 (Fig. 8). However, this needs to be established in a wider sample of tissues to determine if this novel function of MUC4 is tissue specific or mutation specific.

Figure 8
Figure 8
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MUC4; p53; Motility; Invasion; EMT; Lung cancer

© 2013International Association for the Study of Lung Cancer

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