Background
Lung cancer is the leading cause of cancer death in the United States for both men and women. Estimated new cases and deaths for 2016 are projected to 224,390 and 158,080, respectively, and account for nearly 27% of all deaths from cancer [
1]. Malignant mesothelioma is one of the most aggressive forms of cancer with an average survival period of less than one year after diagnosis [
2]. Although smoking rates (a risk factor for lung cancer) have decreased over the years and asbestos (a major cause of mesothelioma) usage in construction has been prohibited, the incidence of lung cancer and mesothelioma is still high, possibly due to their long latency period of development after initial exposure and the complexity and diversity of new carcinogens [
3,
4]. Despite significant advances in treatment management, the prognosis of lung cancer and mesothelioma remains very poor due to limited treatment options and lack of understanding of the disease mechanisms. Thus, identifying the key underlying molecular mechanisms of oncogenesis is essential for early detection and treatment of the diseases.
Mesothelin (MSLN) is a membrane-bound protein with unclear functions. The
Mesothelin gene encodes a 69-kDa precursor protein that is cleaved into a 31-kDa secreted fragment called megakaryocyte potentiating factor (MPF), and a 40-kDa membrane-bound protein termed mesothelin (MSLN), which is a glycoprotein anchored to the plasma membrane by a glycophosphatidyl inositol (GPI) domain [
5,
6]. MSLN is physically undetectable in most normal tissues except mesothelial cells of the peritoneal and pleural cavities and pericardium. However, MSLN is expressed at a high level in almost all mesothelioma and many solid tumors such as in lung cancer (60–70%), pancreatic cancer (80–85%), cholangiocarcinoma (60–65%), ovarian cancer (60–65%), gastric cancer (50–55%), colon cancer (40–45%), breast cancer (25–30%), and endometrial cancer (20–25%) [
7]. Because of its prevalence in cancers, MSLN has recently been targeted for immunotherapy [
7], while the soluble MSLN fragment has been investigated as a biomarker for cancer diagnosis [
8]. Despite extensive studies of MSLN as a potential diagnostic and therapeutic target, neither the physiologic role of MSLN nor its pathological mechanism in cancer is well defined. In lung cancer, accumulating evidence indicates that high expression of MSLN is correlated with poor patient’s overall prognosis and relapse-free survival [
9]. Preclinical studies showed that MSLN is involved in cell proliferation, anoikis resistant and survival [
10‐
12], and its downregulation promotes drug-induced apoptosis and chemosensitivity [
13,
14].
Epithelial to mesenchymal transition (EMT) results in physiological and phenotypic changes where epithelial cells acquire a mesenchymal phenotype. They break down cell-cell and cell-extracellular matrix connections that facilitate their translocation through the extracellular matrix to reach areas of new organ formation. Cancer cells adopt EMT process in the conversion of early stage tumors into dedifferentiated and more malignant states [
15]. EMT plays a crucial role not only in tumor metastasis but also in tumor recurrence [
16‐
18]. The role of MSLN in tumor formation and metastasis of lung cancer and mesothelioma or any role in EMT and cancer stem cell (CSC) regulation is largely unknown.
In this study, we investigated the role of MSLN in lung cancer and mesothelioma by evaluating the effects of MSLN knockdown and overexpression on tumor growth and metastasis in a mouse model. We also assessed the consequences of genetically altered MSLN levels on EMT, the malignant phenotype, and stem properties of human lung carcinoma and mesothelioma cells. Our results demonstrate the essential role of MSLN in promoting EMT and stemness, as well as tumor formation and metastasis.
Methods
Patient tumor samples
Human lung tumor tissues were obtained from the Lung Cancer Biospecimen Resource Network (Charlottesville, VA, USA). Four adenocarcinoma and six squamous cell carcinoma specimens with correlated adjacent healthy tissues were prepared and tested as pairs.
Cell lines and culture conditions
Non-tumorigenic human bronchial epithelial BEAS-2B cells were cultured in bronchial epithelial basal medium along with additives from Lonza Corporation (Walkersville, MD, USA). Human lung carcinoma alveolar epithelial A549 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco, Gaithersburg, MA, USA). Non-small cell lung cancer H460 cells were cultured in RPMI 1640 medium supplemented with 5% FBS and 100 units/ml penicillin/streptomycin. Human pleural mesothelial MeT5A cells were maintained in M199 medium (Life Technologies, Grand Island, NY, USA) with 5% FBS, 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, 1 μg/ml EGF, and 50 μg/ml hydrocortisone. Human pleural mesothelioma H2052 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, and 100 units/ml penicillin/streptomycin. All cells used in this study were obtained from ATCC (Manassas, VA, USA) and were cultured in a humidified atmosphere of 5% CO2 at 37 °C.
Generation of stable MSLN knockdown cell lines
Stable MSLN knockdown lines of H460 and H2052 cells, and their respective vector control lines, were generated via shRNA lentiviral vectors with four shMSLN or scrambled shRNA (OriGene Technologies, Rockville, MD, USA), according to the manufacturer’s instructions. Transfected cells were selected with 1–5 μg/ml of puromycin. Single clones of shMSLN and shRNA controls were verified by Western blotting.
Overexpression of MSLN
MSLN expression plasmid, pAdEasy-MSLN-iCre-HA-Flag (plasmid#31305), was obtained from Addgene (Cambridge, MA, USA). The plasmid was amplified with a DNA midi kit (Qiagen, Hilden, Germany). 2 μg of MSLN plasmid DNA were transiently transfected into Met5A cells with FuGene HD transfection reagent (Promega, Madison, WI, USA). Functional assays were performed 24 h after the transfection.
Cell proliferation
MSLN knockdown and shRNA control cells were seeded at a density of 1.5 × 104 cells per well in 100 μl media in a 96-well plate (Fisher, Waltham, MA, USA). After 24, 48, and 72 h, 20 μl of CellTiter 96 Aqueous One Solution (Promega, Madison, WI, USA) were added to each well and the cells were incubated at 37 °C for an additional 3 h. Viable cells cleaved the reagent’s tetrazolium salt to a soluble formazan dye, resulting in a color change proportional to the number of live cells. Absorbance was measured at 490 nm with a reference wavelength at 630 nm using a BioTek plate reader (BioTek, Winooski, VT, USA).
Cell surface area measurements
Cells were stained with CellTracker™ Green CMFDA dye or CellTracker™ Red CMTPX dye (Thermo Fisher Scientific, Pittsburgh, PA, USA) and seeded into glass chambers at the density of 1 × 10
5/ml. After culturing for 24 h, the cells were fixed with 4% paraformaldehyde and imaged by a Nikon Ti Eclipse fluorescence microscope. The surface area of cells was measured using Image J software (
http://imagej.nih.gov/ij/). A minimum of 200 cells were analyzed for each group.
Control shRNA and shMSLN knockdown cells (2,500 cells) were suspended in 0.5 ml culture medium and mixed with an equal amount of 0.7% agar to a final agar concentration of 0.35%. The mixed cell-agar suspensions were immediately plated onto 6-well plates coated with 0.5% agar in culture medium. Colonies were examined under a light microscope after 2 weeks of culture.
Tumor sphere formation assay was performed under non-adherent and serum-free conditions. Briefly, 5,000 cells were suspended in 0.8% methylcellulose-based serum-free medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 20 ng/ml epidermal growth factor (BD Biosciences, San Jose, CA, USA), 10 ng/ml basic fibroblast growth factor and 5 μg/ml insulin (Sigma-Aldrich, St Louis, MO, USA) in ultra-low adherent 6-well plates (Corning Incorporated, Kennebunk, ME, USA). Cells were cultured for two weeks after which tumor spheres were examined under a light microscope. In order to assess self-renewal property of the cells, spheres were collected by gentle centrifugation, dissociated into single cell suspensions, filtered and cultured under the same conditions to form secondary spheres.
Cell migration and invasion assays
Cell migration was determined by using a 24-well Transwell® unit (Thermo Fisher Scientific, Pittsburgh, PA, USA) with a polyvinylidene difluoride filter (8-μm pore size). Cell invasion was assayed by using a BD Matrigel® invasion chamber (BD Biosciences, Franklin Lakes. NJ, USA). Briefly, 1.5 × 10
4
cells per well (migration) or 3 × 10
4
cells per well (invasion) were seeded into the upper chamber of the Transwell® unit in serum-free medium. The lower chamber was filled with a normal growth medium containing 5% FBS. Chambers were incubated at 37 °C in a 5% CO2 atm for 48 h. Non-migrating or non-invading cells in the inside of the Transwell® inserts were removed with a cotton swab. Cells that migrated or invaded to the underside of the membrane inserts were fixed and stained with Diff-Quik (Dade Behring, Newark, DE, USA). Inserts were visualized and scored under a light microscope (Leica DM, Deerfield, IL, USA). The number of migrating and invading cells from ten random fields were scored.
Pathway specific PCR array
Total RNA from control and MSLN knockdown cells were isolated using a Qiagen RNA mini kit (Qiagen, Valencia, CA, USA) and reverse-transcribed into single stranded cDNA. Differential expression of EMT genes was analyzed using a RT
2 profiler PCR array: EMT Pathway (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. Data analysis was performed online at
www.SABiosciences.com/pcrarraydataanalysis.php.
Immunoblotting
Cells were washed with PBS and lysed on ice with modified RIPA buffer containing protease and phosphatase inhibitor mixtures (Roche Molecular Biochemicals, Indianapolis, IN, USA) for 30 min. The lysates were briefly sonicated and centrifuged at 14,000 × g for 20 min. Cell lysates (40 μg protein) were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The transfer membranes were blocked for 1 h in 5% nonfat dry milk in TBST (25 mM Tris–HCl, pH 7.4, 125 mM NaCl, 0.05% Tween 20) followed by treatment with primary antibodies at 4 °C overnight with gentle shaking. Membranes were washed three times with TBST for 10 min each, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence detection reagents from Millipore (Millipore Corporation, Billerica, MA, USA). Actin was used as a loading control and the data were quantified using Image J densitometry software.
Immunohistochemistry staining
Lung and liver tissue sections in paraffin were deparaffinized and rehydrated. Antigens were retrieved with 10 mM sodium citrate solution in a microwave for 20 min. The slides were then blocked with 3% BSA/0.1% Tween in 1 × PBS blocking buffer for 1 h, and were incubated with anti-human MSLN antibody (Abcam, Cambridge, MA, USA) (1:500) or anti-human mitochondria antibody (EMD Millipore, Temecula, CA, USA) (1:100) overnight at 4 °C. After washing with PBS three times, the slides were incubated with biotinylated secondary antibodies for another hour, followed by ABC reagent (Vector Laboratories, Burlingame, CA, USA) and detected with a DAB kit (Vector Laboratories, Burlingame, CA, USA). After color development, the slides were counterstained with hematoxylin, dehydrated, and mounted with Permount mounting solution. Images were taken using a light microscope with Olympus cellSens Dimension software.
Flow cytometric ALDH activity assay
The AldefluorTM kit (StemCell Technologies, Durham, NC, USA) was used to analyze and isolate the cell population with high ALDH enzymatic activity. Cells were suspended in Aldefluor assay buffer containing ALDH substrate (BODIPY-aminoacetaldehyde, 1 mmol/l per 1x106 cells) and incubated for 40 min at 37 °C. As a control, an aliquot of the sample was treated with 50 mmol/l of the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB).
Tumor xenograft mouse model
Animal care and experimental procedures described in this study were performed in accordance with the Guidelines for Animal Experiments at West Virginia University with the approval of the Institutional Animal Care and Use Committee (IACUC #15-0702). Immunodeficient NOD/SCID gamma mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG; Jackson Laboratory, Bar Harbor, ME, USA), were maintained under pathogen-free conditions within the institutional animal facility. Food and water were given ad libitum. Mice (6/group) were subcutaneously injected with 1 × 106 cells of H460 or H2052 with shMSLN stable knockdown or shRNA control cells suspended in 100 μl of ExtraCel® hydrogel (Advanced BioMatrix, San Diego, CA, USA). Mice were inspected daily for any signs of distress such as weight loss, hunching, failure to groom, and red discharge from the eyes. At the end of the experiments, mice were euthanized and tumors were dissected and weighted. Metastatic nodules were counted from the surface of the intestine, liver, and lungs. Tumor specimens were cut into 5-μm sections and stained with hematoxylin and eosin (H&E) to confirm cancer morphology and metastasis in the organs. All tissue sectioning and staining procedures were performed at the West Virginia University Pathology Laboratory for Translational Medicine.
Statistical analysis
Results are expressed as means ± s.d. from three or more independent experiments to ensure adequate power (>80%). Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by Student’s t test. For all analyses, two-sided P values of ≤ 0.05 were considered statistically significant.
Discussion
Although MSLN appears to be non-essential in normal tissues since MSLN knockout mice exhibit no detectable malfunction in tissue development, reproduction, and blood cell count [
27], clinical studies have shown that high MSLN expression correlates with tumor aggressiveness in many solid tumors [
7]. Consistent with previous studies, our presented results indicate an upregulation of MSLN in human lung tumor tissues and in lung carcinoma and mesothelioma cell lines. Knockdown of MSLN in these cell lines reversed their malignant phenotype as indicated by soft agar colony formation, tumor sphere formation, and cell migration and invasion assays (Figs.
2 and
3) as well as tumor formation in animals (Fig.
5). In non-cancerous cells, overexpression of MSLN promoted a malignant phenotype as indicated by anchorage-independent growth and cell migration and invasion assays (Fig.
4). Together, these results strongly support the general pro-carcinogenic role of MSLN, which is supported by clinical observations that show a linkage between MSLN expression and tumorigenesis in lung, breast, and pancreatic cancers [
9,
19,
28,
29].
Metastasis is the primary cause of death in patients with advanced cancer. In addition to regulating tumor formation, our data also suggest the role of MSLN in controlling metastasis. Supporting this notion knockdown of MSLN effectively inhibited liver and lung metastasis (Fig.
5) and migration and invasion of tumor cells (Fig.
3). Metastasis is a highly complex process, closely associated with EMT, the phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state. This transformation provides cancer cells with the ability to breakdown epithelial cell-cell tight junctions, invade extracellular matrix basement membranes, and enter the circulation to become circulating tumor cells (CTCs). Previous studies showed that CTCs possess both EMT and CSC characteristics [
30,
31]. Cells from primary tumors were found to express a combination of mesenchymal and epithelial markers, whereas CTCs express predominantly mesenchymal markers [
32]. Our study showed for the first time that MSLN regulates EMT, and possibly CTCs and CSCs, which may be responsible for tumorigenesis and metastasis. Lung cancer (H460) and mesothelioma (H2052) cells exhibit some degree of mesenchymal and CTC phenotypes in terms of morphology, basement membrane attachment, and migratory and invasive activities. Knockdown of MSLN dramatically changed the morphology of the cells from a mesenchymal spindle-like shape to epithelial-like shape, increased their adhesion and spreading on cell culture substrata, and decreased their migration and invasion (Fig.
3). These phenotypic changes decreased the likelihood of the cells to exit the tissue and become CTCs, and to metastasize to other tissues.
PCR array and western blot analyses were used to characterize EMT and CSC markers in control and MSLN knockdown cells. The control cells expressed a high level of mesenchymal and CSC markers, whereas the shMSLN cells expressed predominantly epithelial markers (E-cadherin, caveolin, and occludin) and a low level of mesenchymal and CSC markers (Twist, EGFR, Snail, Slug, ABCG2, and ALDH activity (Fig.
6). Recent studies have shown that EMT is a key driver of CSC formation which controls tumor progression and the treatment response [
16,
32,
33]. The low adherent (trypsin sensitive) subpopulation of breast and colon cancer cells exhibited EMT and stem properties with increased ALDH activity [
34]. A cell tracking method demonstrated a dynamic change from EMT, CTCs to CSCs and distant metastasis in vivo in pancreatic cancer [
35]. In mesothelioma cells, we present that knockdown of MSLN reversed the EMT to MET phenotype and significantly reduced CSC markers and ALDH activity, which may contribute to the observed reduction in tumorigenicity and metastasis of the knockdown cells.
MSLN appears to regulate EMT through multiple pathways and downstream targets. For example, knockdown of MSLN promoted the epithelial phenotype by up-regulating E-cadherin, cytokeratins, claudins, occludin, IL1RN, MITF, MSTIR, and NUDT, and by downregulating transcription factors such as Twist, Snail1, as well as fibronectin, ILK, EGFR, and WNT11 (Fig.
6). E-cadherin, encoded by CHD1, is a calcium-dependent cell-cell adhesion glycoprotein. Loss of E-cadherin is associated with mesenchymal transition and metastatic activity of cancer cells [
36]. Claudins and occludin are key components of tight junction proteins, which regulate epithelial/endothelial permeability [
37] and directional migration [
38]. Loss of occludin causes increased cell invasion, reduced adhesion, and impaired tight junction integrity in breast cancer tissues [
39]. Cytokeratins are keratin-containing filaments that preserve cell structure and cell-cell adhesion. Twist is a key transcription factor involved in embryogenesis and development and regulates EMT and cell migration [
40]. Snail belongs to a family of zinc-finger transcription factors that is essential for embryonic development and well-known to induce EMT [
41]. The effect of MSLN on EMT may be cell-line dependent since a previous study by Wang et al. [
11] showed that knockdown of MSLN in H2373 mesothelioma cell line did not affect E-cadherin expression but decreased β-catenin expression and increased Slug expression. Our morphological and functional assays confirmed that knockdown of MSLN in H2052 and H460 cells reversed their EMT phenotypes (Figs.
2 and
3), consistent with our EMT markers expression data (Fig.
6).
Induction of EMT is a highly complex process and involves several coordinated networks and signaling pathways. It is triggered by growth factors, such as transforming growth factor (TGF)-β, fibroblast growth factor (FGF), and epidermal growth factor (EGF). Binding of these growth factors to their respective surface receptors activates intracellular effector molecules and subsequently transcriptional activators such as snail and slug, which regulate functional molecules of EMT [
42]. E-cadherin is a key target of Snail, Twist, and ZEB family members, and is often downregulated in aggressive carcinomas as a result of EMT induction [
43]. Downregulation of E-cadherin weakens cell-cell adhesion, triggers cell migration from the primary tumor to systemic circulation, and promotes CSC formation and metastasis in distant organs [
17,
44]. The impact of MSLN on several EMT and CSC regulatory genes that we have observed suggests that MSLN may act as a master regulator of EMT that controls both local invasion and distant metastasis.
Acknowledgements
Not applicable.