Mesothelin Promotes Invasion and Metastasis in Breast Cancer Cells

Abstract

Objective:

The presence of mesothelin (encoded by the mesothelin [MSLN] gene) in breast cancer is associated with tumour infiltration of the lymph node. This study evaluated whether MSLN overexpression promotes breast cancer cell invasiveness and metastasis.

Methods:

This study evaluated the effects of overexpression of MSLN on extracellular signal-regulated kinase (ERK1/2) and matrix metalloproteinase (MMP)-9 levels, and the invasiveness and angiogenesis of the breast cancer cell line MCF-7 in vitro, and on MCF-7-derived tumour development in vivo.

Results:

MSLN overexpression significantly increased ERK1/2 and MMP-9 protein levels and activity, and the invasive and angiogenic capability of MCF-7 cells, in vitro. Inhibition of ERK1/2 suppressed MMP-9 and the invasive and angiogenic capability of MSLN overexpressing MCF-7 cells. MSLN overexpression also increased MCF-7-derived tumour metastasis in vivo.

Conclusion:

MSLN overexpression promoted the invasive potential of MCF-7 cells through ERK1/2-dependent upregulation of MMP-9; this association may have contributed to metastasis of MCF-7 cells in vivo. Mesothelin may be a useful biomarker for cancer progression and a novel therapeutic or chemopreventive target in human breast cancer.

Introduction

Mesothelin (encoded by the mesothelin, or MSLN, gene) is a differentiation antigen present on normal mesothelial cells of the pleura, peritoneum and pericardium. ¹ The MSLN gene is overexpressed in various cancers including ovarian cancer, pancreatic cancer, adenocarcinoma, mesothelioma, lung adenocarcinoma and acute myeloid leukaemia.^{2 – 5} MSLN overexpression has also been noted in other human cancers such as squamous cell carcinomas of the cervix, lung and head, and in neck carcinomas and endo-metrial adenocarcinomas.^{6 – 8} Immunohisto -chemistry shows infrequent MSLN expression in colorectal, gastric and oesophageal cancers, and an absence of expression in soft tissue sarcomas (with the exception of biphasic synovial sarcomas). ⁶ The apparently limited expression of the MSLN gene in normal human tissues and high levels of expression in several human cancers makes it an attractive candidate for cancer therapy. New therapies, including CAT-5001, MORAb-009 and CRS-207, which target cell surface mesothelin or elicit an immune response against mesothelin, are under investigation in clinical trials.^{9 – 11}

The normal biological function of mesothelin is unclear, although a msln knockout mouse model has shown that, in mice at least, mesothelin is not essential for growth or reproduction. ¹² Silencing of the MSLN gene significantly increases the fraction of cancer cells in S phase and results in significant loss of viability, invasiveness, and morphological alterations in vitro. ¹³

Although the MSLN gene is known to be associated with metastasis and angiogenesis in cancers, the mechanism of action and signalling pathways involved remain unclear. Mesothelin enhances cell invasion by inducing matrix metalloproteinase (MMP)-7 through the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase signalling pathways in ovarian cancer. ¹⁴ MMP-9 plays an important role in the invasion and angiogenesis of cancer cells. In mesothelioma cells, mesothelin promotes tumour invasion and upregulates MMP-9 in MSLN-expressing cells. ¹⁵ The mechanism by which MMP-9 regulates its biological effects in the development of breast cancer is unknown, however.

The presence of mesothelin in breast cancer cells is associated with tumour infiltration of the lymph node. ¹⁶ Additionally, mesothelin can regulate cancer cell invasion via the ERK1/2 signalling pathway, ¹⁴ and MMP-9 is regulated by ERK1/2 in human breast cancer cells. ¹⁷ Mesothelin may, therefore, enhance invasion of breast cancer cells through ERK/MMP-9 pathways.

The present study evaluated the effect of overexpression of the MSLN gene on in vitro invasion and metastasis, via an ERK1/2/MMP-9 pathway, in breast cancer cells, and on in vivo metastasis in breast cancer models.

Materials and methods

Cell Lines and Culture Conditions

The MDA-MB-231 and MCF-7 human breast cancer cell lines were purchased from American Type Tissue Collection (Manassas, VA, USA), and were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 0.1 mmol/l nonessential amino acids, 0.1 mmol/l sodium pyruvate (Life Technologies™, Grand Island, NY, USA) and 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT, USA). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Exponentially growing cells were detached using 0.05% trypsin-ethylenediamine tetra-acetic acid (Life Technologies™) in DMEM for 5 min at 37 °C.

MSLN Variant 1 Gene Plasmid Construction

The full-length cDNA for human the MSLN variant 1 gene (MSLN, Genbank accession no. NM 005823) was amplified by polymerase chain reaction from HeLa cDNA, using the following primers: MSLN forward, 5′-GCCAATCACCCTGCACATCAGAGTT-3″; MSLN reverse, 5′-TTCCCGTTTACTGAGCGC GAGTTCT-3′. The amplified MSLN fragment was then ligated into the pCRII vector (Invitrogen, Carlsbad, CA, USA), released after digestion with the restriction enzymes HindIII and NotI, then religated into the pcDNA3.1 mammalian expression vector containing green fluorescence protein (GFP) (Invitrogen). Plasmids were purified using the Plasmid Midi kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, and the sequence, structure and fidelity of the resulting construct was confirmed by restriction mapping and sequencing (Shanghai Shengong Envirormental Protection Co., Shanghai, China).

Preparation of Transient and Stable MSLN-Transfected MCF-7 Cells

Cells underwent serum starvation for 24 h before transient transfection. For transfection studies, 2 – 4 μg pcDNA3.1-MSLN or pcDNA3.1 control plasmid was transfected into MCF-7 cells (cell density, 1 × 10⁶ per well in a six-well plate) using Lipofectamine^® 2000 (Life Technologies™) according to the manufacturer's instructions. At 48 h after transfection, Western blot analysis was performed, as described below, to check mesothelin protein levels in these transiently transfected cells.

To select stably transfected clones, transiently transfected cell lines positive for mesothelin were cultured in DMEM containing the antibiotic geneticin (G418) (800 μg/ml) for 21 days, then maintained in DMEM containing 400 μg/ml G418.

Treatment of Stable Clones with ERK1/2 and MMP-9 Inhibitors

To study the effects of ERK and MMP, MCF-7 cells (1 × 10⁶ cells per well in a six-well plate) transfected with the MSLN gene (MCF-7-MSLN cells) or the pcDNA3.1 control plasmid (MCF-7-control transfected cells) were cultured without inhibitors or in the presence of 5 μM U0126, an ERK-specific inhibitor (Calbiochem, San Diego, CA, USA), or 2 μM BB94, a nonspecific MMP inhibitor (Shanghai Sangon Biological Engineering Technology & Services, Shanghai, China) for 24 h at 37 °C. Cells were harvested for evaluation of mesothelin, ERK1/2 and MMP-9 protein levels, and MMP-9 proteolytic activity, as described below. After 24 h, the culture conditioned media was also collected and stored at −20 °C

Western Blot Analysis of Mesothelin, ERK1/2 and MMP-9 Protein Levels

Western blot analysis was carried out to determine the levels of mesothelin protein in MDA-MB-231 and MCF-7 cells, and levels of mesothelin, ERK1/2, phosphorylated (p)-ERK and MMP-9 protein in MCF-7-MSLN and MCF-7-control transfected cells.

Cells were harvested by trypsinization, as described above, and lysed overnight at –20 °C in a lysis buffer containing 150 mmol/l NaCl, 1% Triton X-100™, 1 mmol/l phenylmethylsulphonyl fluoride and 25 mmol/l Tris (pH 7.5). Cell debris was sedimented by centrifugation at 12 000 g for 5 min and the protein supernatants were solubilized at 100°C for 5 min in Laemmli sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer containing 100 mmol/l dithiothreitol. The protein concentration of the cell lysates were determined with a protein detection assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions, and 40 μg of each sample was separated by 10% SDS–PAGE at 100 V for 90 min. Separated polypeptides were transferred electrophoretically to 0.2 mm-thick nitro -cellulose membranes (Schleicher and Schuell Bioscience, Keene, NH, USA) at 100 V for 75 min.

Membranes were blocked for 1 h at 4°C in Tris-buffered saline containing Tween 20 (TBS-T; 25 mmol/lTris, pH 8.0, 150 mmol/l NaCl and 0.05% Tween-20) and 5% (w/v) nonfat dried milk. The blots were then probed overnight at 4 °C with the following primary antibodies: mouse antimesothelin (1 : 100 dilution) and rabbit anti-MMP-9 (1 : 100 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-ERK1/2 and p-ERK (1:200 dilution) (Cell Signaling Technology^®, New England Biolabs^®, Ipswich, MA, USA). The blots were washed and developed using species-specific secondary antisera (goat antirabbit immunoglobulin (Ig)G-horseradish peroxidase (HRP) and goat antimouse IgG-HRP; Cell Signaling Technology^®), according to the manufacturer's instructions. Detection of β-actin protein levels, using an anti-β-actin primary antibody (1 : 500 dilution; Santa Cruz Biotechnology), was used as a control for protein loading.

Immunoreactive signals were detected by enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, GE Healthcare Life Sciences, Shanghai, China) according to the manufacturer's instructions. Relative protein levels, normalized using β-actin, were quantified by scanning and pulsed dye laser densitometry (GE Healthcare Life Sciences).

Gelatin Zymography Analysis of MMP-9 Proteolytic Activity

Equal numbers of MCF-7-MSLN and MCF-7-control transfected cells (1 × 10⁶ cells per well in a six-well plate) were grown in culture as described above, in the presence or absence of specific inhibitors, after which conditioned media was collected and concentrated using Amicon filters (Millipore, Billerica, MA, USA) as per the manufacturer's protocol. Proteins concentrated from the conditioned media were electrophoresed (40 μg protein per sample) under nonreducing conditions and the gelatinolytic activity of MMP-9 was determined using a commercial Zymogram Gel Electrophoresis Kit (Invitrogen), designed for the detection and characterization of MMPs, according to the manufacturer's instructions.

Modified Boyden Chamber Assays for Invasion

Invasion assays were performed with stably transfected MCF-7-MSLN and MCF-7-control transfected cells, and untransfected control MCF-7 cells, (1.25 × 10⁴) using a modified Boyden chamber (Micro Chemotaxis Chambers, Shanghai, China) and Matrigel™-coated 8-μm polypropylene filter inserts (Corning Costar, Cambridge, MA) according to the manufacturer's instructions. The degree of cell invasion was determined by counting cells in five microscopic fields per well (× 200 magnification), and the extent of invasion was expressed as the mean number of cells per microscopic field. All experiments were performed in triplicate.

In Vitro Angiogenesis Assay

Human dermal microvascular endothelial cells (HMEC)-1 (Cell research, Shanghai, China) (2.4 × 10⁴ per well) were seeded in eight-well chamber slides and the conditioned medium (1 ml), prepared as described above, was added. Cells were cultured at 37 °C for 72 h until capillary network formation was observed. To quantify the degree of angiogenesis, the slides were stained with haematoxylin and eosin, and the number of branch points and total number of branches per point were counted.

Nude Mouse Metastasis Model

All animal experiments were performed under the approval of the Animal Experimentation Committee of Qingdao University, Qingdao, China. Specific pathogen-free athymic BALB/c female nude mice (age 4 – 6 weeks, weight 18 – 20 g) were housed under a 12-h light/12-h dark cycle with free access to food and water in sterile conditions, in a laminar flow room, in cages with filter bonnets; they were fed a sterilized mouse diet and water.

To induce metastasis, mice (n= 8 per group) were anaesthetized using inhaled sevoflurane and injected with stably transfected MCF-7-MSLN cells, MCF-7-control transfected cells or untransfected MCF-7 cells (1 × 10⁶ cells/mouse in 100 μl of 10 mM phosphate-buffered saline, pH 7.2) into the peritoneal cavity, using a 23-gauge needle. Mice were injected once per day for 20 consecutive days, after which they were sacrificed. Body weight, ascites, and the number and total weight of metastatic tumours were recorded. Tumours were resected in one piece, together with mesenterium and greater omentum, and weighed. In order to minimize the interfusion of blood or tissues, the weight exceeding the mean weight of the mesenterium and greater omentum of normal nude mice of the same age (n = 24) was regarded as the net weight of metastatic tumours. Animal studies were not conducted according to any published national or international protocols or guidelines for animal welfare.

Statistical Analyses

All statistical analyses were performed using the SPSS^® software package, version 10.0 (SPSS Inc., Chicago, IL, USA) for Windows^®. All data were presented as mean ± SE and were analysed using Student's t-test. A P-value < 0.05 was considered to be statistically significant.

Results

Western blot analysis showed that, relative to β-actin, MCF-7 breast cancer cells produced lower levels of endogenous mesothelin protein than MDA-MB-231 cells (0.12 ± 0.02 versus 0.82 ± 0.13). Thus, the MCF-7 cell line was selected for use in MSLN overexpression studies.

The MCF-7-MSLN cells produced significantly higher levels of mesothelin protein levels compared with MCF-7-control transfected cells (1.28 ± 0.16 and 0.14 ± 0.02, respectively; P = 0.003). Intracellular levels of mesothelin in the MCF-7-MSLN cell line were increased 12-fold compared with the parental MCF-7cell line (data not shown).

The number of invasive MCF-7-MSLN cells (204 ± 13) was significantly higher than the number of invasive untransfected control MCF-7 cells (116 ± 23) and MCF-7-control transfected cells (120 ± 20) (P < 0.05).

Compared with MCF-7-control transfected cells, MCF-7-MSLN cells showed significantly higher levels of activated p-ERK protein (0.03 ± 0.00 versus 423 ± 0.07, respectively; P < 0.001) and MMP-9 protein levels (0.12 ± 0.05 versus 0.874 ± 0.23, respectively; P = 0.004). Treating MCF-7-MSLN cells with the specific ERK inhibitor, U0126, resulted in a significant decrease in MMP-9 protein levels (MCF-7-MSLN cells, 0.85 ± 0.18 versus MCF-7-MSLN cells + U0126, 0.08 ± 0.00; P = 0.0024). Gelatin zxymography showed a significant increase in MMP-9 enzymatic activity in MCF-7-MSLN cells (0.524 ± 0.19) compared with MCF-7-control transfected cells (0.08 ± 0.01) (P = 0.036). Treating MCF-7-MSLN cells with U0126 or the nonspecific MMP inhibitor BB94 also reduced their invasive potential in vitro (MCF-7-MSLN cells, 192.4 ± 25.3; MCF-7-MSLN cells + U0126, 95.6 ± 9.4; MCF-7-MSLN cells + BB94, 109.4 ± 10.4).

The mean ± SE number of angiogenic branch points in HMEC-1 cells treated with MCF-7-MSLN cell-conditioned media was 196 ± 23, which was significantly higher than the number of branch points observed in HMEC-1 cells treated with conditioned media from untransfected control MCF-7 (reference 100 ± 0) or MCF-7-control transfected cells (104 ± 20) (P < 0.001). In MCF-7-MSLN cells treated with U0126 or BB94, the mean ± SE number of angiogenic branch points fell to 110 ± 27 and 116 ± 22, respectively, which was significantly lower than observed for untreated MCF-7-MSLN cells (P < 0.001).

Postmortem examination 20 days after cancer cell inoculation found larger peritoneal metastases in the MCF-7-MSLN treatment group (76.4 ± 7.8 mg) compared with the mice injected with MCF-7-control transfected cells (27.3 ± 3.8 mg) or untransfected MCF-7 cells (25.6 ± 5.4 mg) group. In the MCF-7-MSLN group, the number (79.4 ± 13.6; P = 0.023) and the weight of tumours (510.8 ± 46.8 mg; P = 0.038) were significantly increased compared with mice injected with untransfected MCF-7 cells (number, 38.8 ± 4.6; weight, 287.0 mg ± 34.7 mg) or MCF-7-control transfected cells (number, 42.5 ± 3.8; weight, 308.3 mg ± 28.4 mg).

Discussion

Overexpression of MSLN is reported to promote mesothelioma cell invasion. ¹⁵ The role of mesothelin in human breast cancer metastasis is, however, yet to be elucidated. The presence of mesothelin in breast cancer significantly correlates with tumour infiltration in the lymph node and a decreased overall survival rate. ¹⁶ The same study ¹⁶ showed no correlation between the presence of mesothelin and either human epidermal growth factor receptor 2/neu protein, oestrogen receptor or progesterone receptor levels in tumour cells. The overexpression of the MSLN gene promoted the invasiveness, metastasis and angiogenesis of human breast cancer cells in vitro and in vivo in the present study, suggesting that the MSLN gene may be associated with invasion and metastatic spread of cancerous cells during the progression of human breast cancer. Exactly how mesothelin regulates these processes and the signalling pathways involved are, however, yet to be described.

Extracellular signal-regulated kinase 1/2 is an important subfamily of MAPKs that control a broad range of cellular activities and physiological processes. ¹⁸ ERK1/2 can be activated transiently or persistently by the MAPK/ERK kinases MEK1/2, and upstream of MAP3K in conjunction with regulation and involvement of scaffolding proteins and phosphatases. ¹⁸ Tumour growth depends on angiogenesis, and the induction of angiogenesis is one of the most important hallmarks in cancer development. ¹⁹ The activation of ERK can upregulate MMP-9, ¹⁷ which is known to play an important role in the invasion and migration of cancer cells. ²⁰ Regulation of the invasion and migration of MDA-MB-231 cells by the bioactive flavonoid, oxorylin A, involves an ERK1/2-dependent MMP-9 signal. ²¹ Tubular network formation and the migration of MCF-7 cells require MMP-9, which is thought to act through protein kinase C and ERK1/2 signalling pathways. ²² Together, these data suggest that ERK1/2-MMP-9 signalling plays an important role in invasion, migration and angiogenesis of breast cancer.

The present study demonstrated that MSLN overexpression significantly promoted ERK1/2 activity, upregulating MMP-9 and increasing its proteolytic activity. When ERK1/2 activity was inhibited by the specific ERK1/2 inhibitor U0126, the MMP-9 level and activity was inhibited. In migration and angiogenesis assays, MSLN expression activated MCF-7 cell migration and promoted the angiogenesis of HMEC-1 cells in vitro. U0126 and the nonspecific MMP inhibitor BB94 significantly inhibited this mesothelin-associated migration and angiogenesis, suggesting that these events are both ERK1/2 and MMP-9 dependent. In vivo, MSLN-transfected MCF-7 cells significantly increased the number and weight of tumours, indicating that mesothelin-related signalling might play a role in cancer metastases and growth in vivo.

In conclusion, the present study suggests that mesothelin activates cancer cell migration and angiogenesis through the ERK1/2-MMP-9 dependent signalling pathway, as indicated by others. ²² Mesothelin also promoted tumour metastasis in vivo, although the relevant signalling pathways were not investigated. Thus, blocking MSLN may be useful in the treatment of breast cancer.