Decreased MT1-MMP in gastric cancer suppressed cell migration and invasion via regulating MMPs and EMT
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Membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified to play a significant role in several types of cancers, but little is known about the significance of MT1-MMP in gastric cancer patients. The purpose of this study is to investigate the involvement of MT1-MMP in tumor progression of gastric cancer. MT1-MMP expression levels were examined in gastric cancer tissues and cells, and normal gastric tissues and cells. The effects and molecular mechanisms of MT1-MMP expression on cell proliferation, migration, and invasion were also explored. In our results, MT1-MMP messenger RNA (mRNA) and protein expression levels were significantly increased in gastric cancer tissue. Moreover, the overexpression of MT1-MMP was positively associated with the status of clinical stage and lymph node metastasis through real-time PCR. Furthermore, knocking down MT1-MMP expression significantly suppressed the cell migration and invasion in vitro and regulated the expression of MMPs and epithelial-mesenchymal transition (EMT)-associated genes. In conclusions, our study demonstrates that MT1-MMP was overexpressed in gastric cancer tissue, and reduced expression of MT1-MMP suppressed cell migration, invasion, and through regulating the expression of MMPs and the process of EMT in gastric cancer.
KeywordsMT1-MMP Gastric cancer Biomarker Metastasis Epithelial-mesenchymal transition
Gastric cancer is one of the common malignancies worldwide and represents the third leading cause of cancer-related death [1, 2]. Although there has been a steady decline in the incidence and mortality risk of gastric cancer over several decades in most countries [3, 4], the clinical outcome of gastric cancer patients remains unsatisfactory. Most patients present with unresectable or metastatic disease at the time of diagnosis . Thus, development of novel diagnostic and therapeutic approaches for gastric cancer is urgently needed.
Membrane type 1-matrix metalloproteinase (MMP) (MT1-MMP), which is a member of the MMPs family, has been implicated in multiple biological processes for its extracellular matrix degrading and accelerating angiogenesis . Present studies indicated that a selective MT1-MMP inhibitor reduced cancer cell motility and tumor growth in human melanoma, fibrosarcoma, tongue squamous cell carcinoma, oral carcinoma, and breast carcinoma cell line . Furthermore, increased plasma membrane localization of MT1-MMP can facilitate prostate cancer cell invasion and metastasis . Interestingly, recent studies showed that upregulation of MT1-MMP was associated with the transcriptional changes during tumor formation because gene mutations have not been observed for MT1-MMP in cancer . MicroRNAs, an abundant class of small and non-coding RNAs, are significantly regulating factor for MT1-MMP. In lung cancer and esophageal cancer, MT1-MMP was a functional target of miR-133a and that miR-133a suppresses proliferation, migration, and invasion by targeting MT1-MMP [8, 9].
In this context, MT1-MMP has been implied to involve in the development and progression of human cancer, but the accurate role of MT1-MMP in gastric cancer remains unknown. The aim of this study was to identify the pathological roles of MT1-MMP in gastric cancer.
Materials and methods
MKN-28 (well differentiated), NCI-N87 (well differentiated), MKN-74 (moderately differentiated), SGC-7901 (moderately differentiated), MKN-45 (poorly differentiated), AGS (poorly differentiated), and GES-1 (a normal gastric epithelial cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified CO2 (5 %) atmosphere.
Collection of clinical specimens
Forty-six fresh gastric cancer tissues and 23 fresh adjacent normal tissues were obtained at the time of diagnosis before any therapy from the First Affiliated Hospital of Sun Yat-sen University. Adjacent non-tumor tissues were located more than 5 cm away from the tumor tissues and verified by pathological examination. All fresh samples were obtained from surgical biopsy and immediately preserved in liquid nitrogen. The clinical processes were approved by the Ethics Committees of the First Affiliated Hospital of Sun Yat-sen University. The patients provided informed consents. The clinical staging was based on the seventh AJCC Cancer Staging Manual.
To quantitate messenger RNA (mRNA) expression, total RNA was extracted from clinical samples with RNAiso Plus (TaKaRa, Japan). The isolated total RNA was reverse transcribed using the PrimeScript RT Master Mix (Perfect Real Time) (TaKaRa, Japan) for MT1-MMP, according to the manufacturer’s instructions. Relative expression was calculated via the comparative cycle threshold (Ct) method and was normalized to the expression of GAPDH. The sequence-specific forward and reverse primers sequences for MT1-MMP mRNA were 5′-GGATACCCAATGCCCATTGGCCA-3′ and 5′-CCATTGGGCATCCAGAAGAGAGC-3′, respectively. Forward and reverse primers sequences for GAPDH mRNA were 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′-TGGTGAAGACGCCAGTGGA-3′, respectively. qPCR was performed using SYBR Premix Ex TaqTM II (TaKaRa, Japan) on a LightCycler (Roche Diagnostics, USA). Relative quantification of miRNA expression was calculated by using the 2−△△Ct method. The raw data were presented as the relative quantity of target mRNA, normalized with GAPDH, and relative to a calibrator sample. All qRT-PCR reactions were performed in triplicate.
MT1-MMP small interfering RNA (siRNA) (si-MT1-MMP) and non-targeting siRNA (si-control) were purchased from GenePharma (Shanghai, China) and used at 20 mM. Opti-MEM transfection media and Lipo2000 (both from Invitrogen, USA) were used to transfect the cells once they reached 60 % confluency. Knockdown was assessed by Western blotting after 48 h of transfection.
Cell proliferation assays
Cell proliferation was analyzed using MTT assay. Briefly, 1 × 103 cells were seeded into a 96-well plate with quadruplicate repeat for each condition. For si-MT1-MMP and si-control, the cells were incubated for 1, 2, 3, and 4 days. Twenty microliters of MTT (5 mg/ml) (Sigma, USA) was added to each well and incubated for 4 h. At the end of incubation, the supernatants were removed and 150 μl of DMSO (Sigma, USA) was added to each well. The absorbance value (OD) of each well was measured at 490 nm. Experiments were performed three times.
Cell migration and invasion assays
In vitro cell migration and invasion assays were examined according to previous study . Briefly, 1 × 105 cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning, Corning, USA). After the cells were incubated for 12 h, Giemsa-stained cells adhering to the lower surface were counted under a microscope in five predetermined fields (×100). For the cell invasion assay, the procedure was similar to the cell migration assay, except that the transwell membranes were pre-coated with 24 mg/ml Matrigel (Corning, USA).
Western blot was carried out according as described  with anti-MT1-MMP antibody (1:1000; Chemicon, USA); anti-MMP1, MMP3, MMP7, MMP8, MMP10, MMP11, and MMP13 (1:1500, Abcam, USA); and anti-MMP2, MMP9, Zo-1, E-cadherin, Vimentin, Snail, Slug, and ZEB1 (1:1000; Cell Signaling Technology, USA). An HRP-conjugated anti-rabbit/mouse IgG antibody was used as the secondary antibody (1:2000; Cell Signaling Technology, USA). Signals were detected using enhanced chemiluminescence reagents (Pierce, USA).
All data were analyzed for statistical significance using SPSS 13.0 software. Two-tailed Student’s t test was used for comparisons of two independent groups. One-way ANOVA was used to determine cell growth in vitro. A P value of less than 0.05 was considered statistically significant.
MT1-MMP is expressed at high levels in gastric cancer
High expression of MT1-MMP was associated with gastric cancer progression
We next analyzed the correlation between the expression of MT1-MMP and clinicopathological characteristics of gastric cancer patients. MT1-MMP was associated significantly with clinical stage (I–II vs. III–IV, P < 0.001) and lymph node metastasis (absence vs. presence, P = 0.004) (Fig. 1c–d).
Expression of MT1-MMP in gastric cancer cell lines
We first analyzed the expression level of MT1-MMP in a panel of gastric cell lines with different degrees of differentiation including MKN-28 (well differentiated), NCI-N87 (well differentiated), MKN-74 (moderately differentiated), SGC-7901 (moderately differentiated), MKN-45 (poorly differentiated), AGS (poorly differentiated), and GES-1 (normal gastric epithelial cell line). We observed that MT1-MMP expression was relatively higher in poorly differentiated cells compared with normal gastric epithelial cell and well-differentiated cells (Fig. 1e), suggesting that MT1-MMP expression may be associated with the degree of gastric cell differentiation. Based on this expression pattern, we therefore chose SGC-7901 and AGS cells for the following loss-of-function studies.
Reduced MT1-MMP expression has no effect on the proliferation of gastric cancer cells
Knockdown of MT1-MMP inhibits the cell migration and invasion
To examine the effect of MT1-MMP on cell migration, after 24 h of transfection, the percentage of migrated cells in both si-MT1-MMP SGC-7901 and AGS cell groups was significantly less than that in the si-control cells (for both P < 0.001, Fig. 2b). Using a Boyden chamber coated with matrigel, we determined changes in cell invasiveness after 24 h of transfection. Compared with the si-control cells, si-MT1-MMP SGC-7901 and AGS cells both showed significantly decreased invasiveness (both P < 0.001, Fig. 2c).
MT1-MMP controls the expression of metastasis-associated and EMT-associated genes in gastric cancer
MT1-MMP, the first member of membrane-type MMPs family, is distinguished from soluble MMPs by a C-terminal transmembrane domain and a cytoplasmic tail . In the human genome, MT1-MMP is encoded by a single copy gene located on chromosome 14 . MT1-MMP has been suggested to involve in many biological processes, such as proliferation, invasion, angiogenesis, and basement membrane remodeling .
The MT1-MMP overexpression was also observed in several types of human cancer, such as lung cancer [12, 13], prostate cancer [14, 15], and glioblastomas , but little is known about the role of MT1-MMP in gastric cancer patients. In our study, we found that mRNA and protein levels of MT-1MMP were increased in gastric cancer tissues. In addition, we firstly found MT1-MMP expression was relatively higher in poorly differentiated cells compared with normal gastric epithelial cell and well-differentiated cells, suggesting that MT1-MMP expression may be associated with the degree of gastric cell differentiation. Furthermore, our study suggested that MT1-MMP overexpression was positively correlated with clinical stages and lymph node metastasis in patients with gastric cancer. Similarly, Zhou et al.’s  and Wang et al.’s  reports indicated that high MT1-MMP protein level significantly correlated with lymph node metastasis and number of metastatic lymph nodes in patient with non-small cell lung cancer. Moreover, MT1-MMP overexpression has been shown to be an independent poor prognostic factor in several types of human cancers such as gastric cancer [18, 19], breast cancer , glioma , colorectal cancer , ovarian cancer , and prostate cancer . A meta-analysis, which included 1918 cases from 11 studies, showed that MT1-MMP is a potential prognostic factor in human cancers, and the pooled hazard ratio (HR) and corresponding 95 % confidence interval (CI) was 2.46 (95 % CI 1.75–3.47) .
Present studies indicated that a selective MT1-MMP inhibitor reduced cancer cell motility and tumor growth in human melanoma, fibrosarcoma, tongue squamous cell carcinoma, oral carcinoma, and breast carcinoma cell line . Furthermore, increased plasma membrane localization of MT1-MMP can facilitate prostate cancer cell invasion and metastasis . Our in vitro experiments indicated that knocking down MT1-MMP expression had no effect on gastric cell proliferation, but significantly suppressed the cell migration and invasion. MT1-MMP was originally known as a tumor-specific activator of MMP2 [26, 27] and is now identified to activate MMP13 and degrade a variety of ECM components, including fibronectin, collagens, and laminins . In addition, the expression of MT1-MMP also showed a significant positive correlation with the expression of MMP1, MMP2, MMP3, MMP9, MMP11, and MMP13 from an analysis of a published database . In order to identify and understand the molecular mechanisms of MT1-MMP in gastric cancer progression, we measured the effect of knocking down MT1-MMP on other types of MMPs. We found that decreased level of MT1-MMP inhibited the expression of MMP2, MMP9, and MMP13, which was consistent with previous studies in other types of cancer [26, 27, 30, 31]. Moreover, MT1-MMP activity is not restricted to extracellular matrix degradation and is necessary for induction of the epithelial-mesenchymal transition (EMT) .
EMT is a critical process by which epithelial cells lose their epithelial morphology and acquire a mesenchymal phenotype, characterized by the decrease of epithelial proteins such as E-cadherin and ZO-1, and the increase of mesenchymal proteins such as vimentin and fibronectin. It is widely accepted that EMT plays a significant role during tumor invasion and metastasis, and aggressive cancer cells often present with a loss of epithelial characteristics and acquire a mesenchymal phenotype. Snail, slug, and ZEB1 play a central transcriptional role in the regulation of EMT. In oral squamous cell carcinoma, Yang et al. indicated that overexpression of MT1-MMP induces EMT and results in the acquisition of cancer stem cell-like properties . Similar to our study in gastric cancer, we found downregulation of MT1-MMP markedly inhibited the progress of EMT. These results consistently suggested that MT1-MMP play an important role in regulating MMPs and EMT.
In summary, our studies suggested that the expression of MT1-MMP is significantly increased in gastric cancer cell lines and clinical sample and correlated with clinical stage and lymph node metastasis of gastric cancer patients. Furthermore, the MT1-MMP expression significantly regulated the cell migration and invasion in vitro and the expression of MMPs and EMT-associated genes.
Conflicts of interest
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