MENA promotes esophageal squamous cell carcinoma cell invasion and migration via MMP-2, MMP-9 and AKT activation

Background: Esophageal squamous cell carcinoma (ESCC) is a fatal disease with poor prognosis. The predominant reason for ESCC-related death is metastasis caused by tumor cell invasion. Human MENA protein is a member of Ena/Vasp family, which plays a critical role during tumor cell invasion. However, the biological effect of MENA in ESCC cell lines remains unclear Methods: In this study, fluorescent quantitative real-time PCR (qRT-PCR) were conducted to detect the mRNA expression of MENA in tumor and para-cancer tissue, CCK-8 assay and clone formation assay were conducted to evaluate cell proliferation activity, Transwell assay and wound-healing assay were conducted to detect the changes of cell invasion and migration capacity, siRNA and MENA expression vector were constructed to explore biological function of MENA in ESCC cell lines. Western blot analysis were conducted to detect the expressions of MENA , molecular markers of epithelial-mesenchymal transition (EMT), Akt, p-Akt, MMP-2 and MMP-9 respectively in ESCC cell line. Results: The qRT-PCR experiment results showed that MENA expression in ESCC tissue of 35 patients was relatively higher than that in tissue adjacent to cancer. CCK-8 assay suggested that tumor cell proliferation capacity was suppressed followed by the knockdown of MENA expression in Mena high ESCC cell TE13 and was potentiated by the overexpression of MENA in Mena low ESCC cell TE1. Transwell assay and wound healing assay demonstrated that interfering in MENA could inhibit TE13 cells invasion and migration capacity by affecting the expressions of Matrix metalloproteinase-2(MMP-2) and Matrix metalloproteinase-9 (MMP-9), in contrast, overexpression of MENA in Mena low ESCC cell TE1 could promote invasion and migration by up-regulated expression of MMP-2 and MMP-9. Western blot analysis indicated that interfering of MENA expression could affect EMT-related molecular markers (E-cadherin, N-cadherin, Snail, Slug), Akt and p-Akt Conclusions: Our study reveal that MENA could promote

Esophageal cancer is one of the most common malignant tumor in the digestive system [1], East Asia is the region with the highest incidence and mortality of esophageal cancer [2]. Esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) is the two main histological type of EC, EAC is more common in the developed countries compared with ESCC, which is mainly occurring in Asia, especially in China. [3][4][5][6]. At present, ESCC is the main histological subtype which account for more than 90% of cases of all esophageal cancers, and the 5-year survival rate of ESCC patient is still quite poor [7,8]. Most critically, nearly 90% of ESCC patients are in the advanced stage at the time of diagnosis and more than half of them exhibit the local invasion and metastasis, which is the leading cause of death of ESCC [9,10]. Although there is new progress in therapeutic strategies, a large number of patients with ESCC remain poor clinical outcome. Therefore, it is particularly necessary to seek the novel prognostic biomarker for metastasis and molecular therapeutic target.
Human MENA encoded by the ENAH gene is a member of the ENA/VASP family of actin filament elongation factors [11,12] Recent studies found that MENA was highly expressed in a variety of human cancers, such as breast cancer, non-small cell lung cancer, malignant melanoma, and pancreatic cancer. [13,14]. In addition, it shows that high expression of MENA is highly correlated with tumor metastasis and malignancy [15,16] Real-time qRT-PCR Quantitative real-time RT-PCR (real-time qPCR) assay was performed as described previously [17].
Briefly, total RNA was extracted from ESCC tumor tissue and and paired adjacent normal tissue by using TRIzol Reagent (Tiangen, Beijing, China). Target genes were amplified by QuantStudio™ 3 Real-Time PCR System(Thermo Fisher Scientific, Carlsbad, CA) with the following specific primers: MENA forward 5'-TCAAGGGTAAGGGAAACTGG-3', and reverse 5'-TGGCTCACAAGTGGTCCTCC-3'; GAPDH forward 5'-CTCCTCCTGTTCGACAGTCAGC-3', and reverse 5'-CCCAATACGACCAAATCCGTT-3'. The data analysis was conducted using the comparative threshold cycle method (ΔΔCt) in which all MENA Ct values in the ESCC tumor tissue were first normalized to GAPDH Protein extraction and Western blotting Western blotting assay was performed as our previous report [17]. Briefly, Cells were washed twice with ice-cold PBS, and collected cells were lysed in buffer (Solarbio, Beijing, China). Protein

Colony formation assay
Cells were trypsinized after finishing with siRNA or MENA expression plasmid transfection, resuspended cells with PBS and calculate. 500 cells were seeded in a signal well of a six-well-plate and cultured at 37ºC in an atmosphere of 5% CO2 for 10 days. Colonies were washed twice with PBS, and fixed with 4% paraformaldehyde solution for 20 min. After fixation, 0.1% crystal violet was added for staining for 30 min. After being left to dry at room temperature, photographs were recorded, and three replicate experiments were set for each group. Colony numbers were analyzed using Image J. 7

Migration and invasion assays
To determine whether the invasion and migration ability of ESCC cells was mediated by siRNA and MENA expression plasmid. Transwell assay was performed in Transwell chambers (8- ESCC tumor tissue expressed higher MENA mRNA than paired normal adjacent tissue (Fig. 1C), and it can be seen that the data distribution of the adjacent tissues is relatively concentrated, while the data distribution of the tumor tissues is relatively discrete, which reflects the high heterogeneity of the tumor tissues to some extent.

Expression of MENA in a panel of ESCC cell lines
We detected MENA protein expression in a series of ESCC cell lines as well as normal esophageal epithelial cell by Western blotting. We found that MENA protein expression was significantly higher than normal esophageal epithelial cell Het-1A which is consistent with results of qPCR. However, MENA expression was different among these ESCC cell lines, TE13 cells exhibited the highest MENA expression than other ESCC cell lines, especially in TE1 which is even harbored equal expression of MENA compared with Het-1A (Fig. 1A). Thus, we compared TE1 and TE13 in cell proliferation by CCK-8 assay, colony formation by colony formation assay and cell invasion and migration by transwell assay and wound-healing assay, TE13 showed much stronger capabilities of cell proliferation (Fig. 2C), colony formation( Fig. 2A and B), cell invasion and migration (Fig. 2D, E, and F). Therefore, to further explore the functional roles of MENA in ESCC cells, TE13 with the highest MENA level and TE1 with the lowest MENA level were employed for further investigation.

MENA mediates ESCC cells proliferation and colony formation
We silenced MENA expression in TE13 cells with MENA specific siRNA in order to evaluate whether MENA could mediate ESCC cell proliferation and colony formation. Western blotting results shows that MENA expression was significantly suppressed after MENA-siRNA transfection in TE13 cells (Fig. 3A).
Cell proliferation and colony formation were suppressed in siMENA group, compared with siNC group (Fig. 3B, C, D). Since MENA protein expression was much lower in TE1 cells compared with TE13 cells ( Fig. 1A and B), therefore, we constructed an MENA expression vector (pGCMV) and transfected TE1 cells with it to further confirm the effect of MENA in ESCC cells proliferation and colony formation (Fig. 4A). The cell growth rate and colony formation were enhanced in TE1 cells transfected with 9 MENA expression plasmid compared with TE1 cells transfected with empty vector. (Fig. 4B, C, D).
These results indicate that MENA could mediate cell proliferation and colony formation.

MENA promotes cell migration and invasion in ESCC cells
Wound healing assay revealed that migration area of TE13 cells in MENA-siRNA group was all lower after 24 hours than those in siNC group (Fig. 3G). However, migration area of TE1 cells transfected with MENA expression plasmid is higher after 24 hours than those in control group (Fig. 4G).
Meanwhile, We also conducted transwell assay to evaluate the effects of MENA expression on ESCC cell migration and invasion. Silencing MENA expression in TE13 cells significantly decreased the number of cells that migrated or invaded through the membrane in the transwell chamber (Fig. 3E, F).
Conversely, overexpression of MENA enhanced the migration and invasion of TE1 cells compared with control group (Fig. 4E, F). These results suggest that the expression level of MENA strongly correlated with the ESCC cell migration and invasion, therefore increased MENA expression may promote ESCC invasion and tumor metastasis.

Over expression of MENA promotes ESCC cell EMT
Epithelial-mesenchymal transition plays important role during tumor cell invasion and metastasis [18,19], this biological process is mainly manifested as down-regulated expression of E-cadherin, substrate degradation, and up-regulated expressions of N-cadherin and two EMT inducer Snail and Slug [20]. In order to study whether MENA could affect ESCC cell EMT, we explored the EMT specific markers via western blotting. We found that after TE1 cells was transfected with MENA expression vector EMT-related molecular markers significantly changed, E-Cadherin was down-regulated, N-Cadherin and two EMT transcription factor Snail and Slug were up-regulated ( Fig. 5A and B). In TE13 cells, Snail, Slug and mesenchymal marker N-Cadherin were down-regulated in MENA-siRNA group compared with siNC group, however E-Cadherin shows no difference between hMENA-siRNA group and siNC group. Based on the EMT specific markers expression of above, we suggest that MENA plays a role during ESCC cell perform EMT.

MENA expression correlates with MMP-2 and MMP-9 expression in ESCC cells
Strong correlation between MENA and cell invasion was fully demonstrated with MENA knock-down in TE13 cells and MENA over expression in TE1 cells, and given that Matrix metalloproteinases(MMP) is highly required during tumor cell invasion [21]. We conducted Western blotting and Immunofluorescence to further confirm whether knock-down and over expression of MENA could affect the expressions of MMP-2 and MMP-9 proteins, which are considered as critical player in the process of tumor invasion and metastasis [22,23]. We found that MENA-siRNA significantly downregulated MMP-2, MMP-9 expressions, compared with siNC group (Fig. 6A and B)., the Immunofluorescence showed that MENA-siRNA group gave weaker fluorescence signal of MMP-9 than siNC group (Fig. 6C), which was consistent with the outcomes from Western blotting. Conversely, Western blotting experiment shows that TE1 cells transfected with MENA expression vector showed higher expression of MMP-2 and MMP-9 compared with TE1 cells transfected with control plasmid (Fig. 6A, B). These findings suggest that MENA mediated cell invasion may be achieved by degrading and up-regulating of MMP-2 and MMP-9 expression.

MENA expression affects AKT signaling pathway
Akt signaling pathway is critical in tumor genesis and plays important role during cell proliferation [24]. Due to knock-down MENA strongly inhibited cell proliferation and cell colony formation in TE13 cells and over expression of MENA in TE1 cells gave the opposite outcomes. In line with these results, we observed decreased Akt and phosphorylated Akt expression in MENA-siRNA group compared with siNC group via western blotting (Fig. 6A, B), and over expression of MENA gave increased Akt and phosphorylated Akt expression (Fig. 6A, B). In summary, the results implied MENA affect Akt signaling pathway in ESCC cells

Discussion
Tumor metastasis is the most dangerous process in the development of tumors. According to statistics, only about 10% of cancer related mortality are caused by primary tumors, whereas metastatic tumor account for 90% of cancer related mortality [25]. The high metastasis rate of esophageal squamous cell carcinoma has always been the main reason for the high mortality rate of this cancer. How to predict and suppress the metastasis of the tumor as soon as possible is a major problem in the treatment of esophageal squamous cell carcinoma.
In the current study, we found mRNA expression of MENA were significantly higher in primary ESCC tissues compared with adjacent non-tumor tissues, and interestingly, qPCR analysis shows that data distribution of the adjacent tissues is relatively concentrated, while the data distribution of the tumor tissues is relatively discrete, this reflects the high heterogeneity of tumor tissues. The MENA expression was remarkably higher in ESCC cell lines than it in normal human esophagus epithelial cell Het-1A which is consistent with the qPCR results from ESCC patient tissue. We noticed that expression of MENA in TE13 is significantly higher than other ESCC cell lines while expression of MENA in TE1 is the lowest among ESCC cell lines. We then compared TE1 and TE13 in proliferation, colony formation, invasion and migration in vitro, and we found that TE13 is much stronger than TE1 in proliferation, colony formation, invasion and migration.
To better explore the role of MENA in ESCC cell lines, we knocked down MENA expression in TE13 cells and over-expressed MENA in TE1 cells, we found that silencing MENA expression in TE13 cells significantly suppressed cell proliferation and colony formation, whereas MENA overexpression in TE1 cells enhanced cell proliferation and colony formation. These results indicated that MENA may promote tumor cell growth.
Previous studies have shown that MENA proteins localize to focal adhesions, the leading edge of lamellipodia, and the tips of filopodia, and it helps tumor cells to form lamellipodia and filopodia which facilities ECM degradation [26]. Frank B Gertler an etc identified MENA as regulator of carcinoma cell invasion and plays a critical role during breast cancer metastasis [27]. Therefore, In our study, we found silencing MENA expression in TE13 remarkably inhibited cell invasion and migration, in siMENA group almost no cell invaded into the lower chamber whereas the siNC group exhibited much stronger capability of invasion and migration. TE1 cells gained higher capabilities of invasion and migration after transfected with MENA expression vector.
The initial step in carcinoma cell invasion is form invadopodia, which protrude into the matrix and secrete proteases focally that degrade the matrix [28]. The acquired invasion ability of carcinoma cells is often supported by their secretion of matrix metalloproteases (MMPs) [29]. In current study, We found that knock down MENA expression inhibited MMP-2 and MMP-9 secretion while over expressed MENA promote MMP-2 and MMP-9 secretion. Furthermore, Immunofluorescence showed 12 that weaker fluorescent signal in siMENA group compared to siNC group. We also found that interference MENA expression in ESCC cell lines could affect Akt signaling pathway which plays critical roles in tumor cell proliferation, migration and invasion.
Epithelial-mesenchymal transition in cells often enhances the cell's ability to invade and migrate. EMT occurs as a decrease in the expression of E-cadherin and an increase in the expression of N-cadherin and EMT inducer such as snail and slug [30,31]. High expression of E-cadherin will increase the adhesion between cells, while high expression of N-cadherin and Vimentin will make the cells lose adhesion and promote cell movement [32]. Our study showed that knocked down MEAN expression MENA has different isoform due to alternative splicing, including the MENA INV and MENA 11a [35,36].
MENA INV is highly expressed specifically in aggressive tumors and has a very strong pro-invasion effect. Recent studies show that MENA INV can promote formation of cell membrane protrusion which 13 is induced by epidermal growth factor both in vivo and in vitro, and it can enhance the ability of tumor cells to degrade extracellular matrix, thereby promoting the migration and invasion of tumor cells [37,38]. However, MENA 11a is specifically expressed in tumor tissues with low invasion and high adhesion. The expression of MENA 11a in orthotopic tumors tissue is significantly higher than MENA INV , and the epithelial-like tumor cells also show higher expression of MENA 11a than mesenchymal-like tumor cells in breast cancer [39,40] The study was approved and supervised by the research ethics committee of Zhengzhou University, Zhengzhou, China. All procedures performed in studies were in accordance with the ethical standards.   Immunofluorescence assay detects weaker fluorescence signal in siMENA group compared to siNC group.

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