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Aberrant methylation of EYA4 promotes epithelial‐mesenchymal transition in esophageal squamous cell carcinoma

EYA4, one of the four members of the EYA gene family, is associated with several human cancers. However, its biological functions and molecular mechanisms in the progression of cancer, particularly in esophageal squamous cell carcinoma (ESCC), remain unknown. In the present study, we found that EYA4 was underexpressed and hypermethylated in most of the ESCC cell lines tested (85.7%, 6/7). Treatment with 5‐aza‐dC and/or trichostatin A (TSA) restored EYA4 expression in ESCC cell lines, which indicates that EYA4 expression was epigenetically regulated. Similarly, EYA4 was aberrantly hypermethylated in ESCC tissues (78%, 39/50) and downregulation of EYA4 occurred in approximately 65% of primary ESCC at protein level where it was associated significantly with TNM stage and lymph node metastases. Knockdown of EYA4 in KYSE30 and KYSE70 ESCC cells using small hairpin RNA increased migration and invasive motility in vitro. Conversely, the overexpression of EYA4 in KYSE180 and KYSE450 promoted an epithelial phenotype, which consisted of decreased migration and invasion abilities and a decrease in TGF‐β1‐induced epithelial‐mesenchymal transition. Mechanistically, EYA4 overexpression reduced the phosphorylation of Akt and glycogen synthase kinase (GSK) 3β, which led to the inactivation of slug. In addition, we found that TGF‐β1 decreased EYA4 expression in both a dose‐dependent and a time‐dependent manner in KYSE30 cells, accompanied by an increase in the expression of DNA methyltransferases, especially DNMT3A. In summary, EYA4 is frequently hypermethylated in ESCC and may function as a tumor suppressor gene in the development of ESCC.

The eyes absent (Eya) gene family, which was initially identified in Drosophila, is a key regulator of eye development. 4 Four EYA proteins (EYA1-4) are encoded in the mammalian genome and are characterized by a highly conserved C-terminal domain (ED) and a poorly conserved N-terminal domain (NTD). The ED domain is essential for mediating the interaction with other proteins, such as Six and Dach family proteins. 5,6 The NTD domain contains a proline-serine-threonine-rich region and functions as a transcriptional activator. 7,8 Recently, accumulating evidence has shown that EYA functions in the mediation of DNA repair, cell apoptosis, angiogenesis and tumor growth. [9][10][11][12][13] EYA4 was found to be epigenetically silenced and was reported to function as a potential tumor suppressor gene in various types of common human cancers such as esophageal adenocarcinoma, 14 breast cancer, 15 lung cancer 16 as well as in oral dysplasia. 17 In contrast, EYA4 has been reported to play an oncogenic role in peripheral nerve sheath tumors and breast cancer. 13

| Drug treatment
Cells were seeded in 10-cm culture dishes for 24 hours and then treated with 10 lmol/L 5 0 -aza-2 0 -deoxycytidine (5-aza-dC, Sigma-Aldrich, St Louis, MO, USA) for 72 hours followed by treatment with 0.25 lmol/L TSA (Sigma-Aldrich, St Louis, MO, USA) 12 hours. For the 5-aza-dC or TSA treatment, the cells were exposed to 10 lmol/L 5-aza-dC for 72 hours or DMSO for 72 hours followed by incubation with 0.25 lmol/L TSA for 12 hours. Culture medium with drugs were refreshed every 24 hours. TGF-b1 treatment was performed at the indicated concentration (Peprotech, Rocky Hill, NJ, USA).

| DNA extraction, bisulfite modification, methylation-specific PCR and bisulfite sequencing
Genomic DNA was isolated using QIAamp DNA Mini Kits (Qiagen, Hilden, Germany), 500 ng of genomic DNA was then subjected to bisulfite conversion using EZ DNA Methylation Kits (Zymo Research, Orange, CA). MSP was performed with specific primers (listed in Table S1) for either methylated or unmethylated DNA, as previously described. 19 Bisulfite sequencing was performed by amplifying bisulfite-treated genomic DNA of EYA4 promoter region by using primers containing no CpG site (see Table S1). The PCR products were cloned into pGM-T cloning vector (Tiagen, Beijing, China) and sequenced.

| RNA isolation and quantitative reverse transcription-PCR
Total RNA was isolated from cell lines and tissues using TRIzol Reagent (ThermoFisher, Rockford, USA) according to the manufacturer's protocol. Reverse transcription was performed with 1 lg of total RNA using RevertAid First Strand cDNA Synthesis Kits (Ther-moFisher, Rockford, USA). EYA4 cDNA were quantified using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA).
The sequences of the primers used for real-time PCR are shown in Table S1.

| Plasmid construction and transfection
The sequences of 2 human shRNA used to repress EYA4 expression are shown in Table S2. All the shRNA were cloned into the pLKO.  The invasion assay was performed in the same manner as the migration assayexceptMatrigelTranswells(CorningCostar,USA)wereused.
For the drug treatment, cells were preincubated with TGF-b1 (5 ng/mL) or LY294002 (20 lmol/L, Cell Signaling Technology, USA) for 24 hours in a CO 2 incubator. Then the Transwell assay was performed as described above.

| Immunofluorescence
Cells were cultured in a 96-well plate, fixed in formaldehyde, permeabilized with 0.25% Triton X-100, and incubated with an EYA4 antibody LUO ET AL. 2.10 | Cell proliferation assay and determination of the IC50 by WST-8 assay Cell proliferation was examined using a CCK8 assay (Dojindo, Kumamoto, Japan).
KYSE30 were cultured in 96-well (1000 cells/well) for 5 days. The absorbance at 450 nm was measured by a microplate reader.
KYSE30 were cultured in 96-well plates (1500 cells/well) overnight and then treated with serial dilutions of cisplatin (1-10 lmol/ L). The cells were incubated for 72 hours, and CCK8 assays were performed as described above to determine the IC50. The IC50 was calculated by GraphPad Prism v5.0 (GraphPad Software). Experiments were repeated in triplicate.

| Immunohistochemistry
Briefly, tissue sections were de-waxed, rehydrated and incubated in retrieval buffer solution for antigen recovery. The DAB Kit (Zsgb-bio, Beijing, China) was used to visualize protein expression after staining with anti-EYA4 (1:50 dilution). The intensity of immunohistochemistry (IHC) staining in the tumor cells was analyzed independently by 2 pathologists.

| Statistical analysis
Data are presented as the means AE standard error of the mean (SEM). To evaluate significant differences between 2 matched pair   Figure 2B). Similarly, we detected a significantly higher density of methylated CpG sites in primary ESCC tissues compared with adjacent non-tumor tissues ( Figure 2C). Next, we integrated the methylation data with paired mRNA expression data from ESCC tissue samples and found that samples with EYA4 hypermethylation were more likely to have reduced EYA4 expression than samples without EYA4 methylation ( Figure 2D). The association between EYA4 methylation and the clinicopathological features of ESCC was also analyzed. No significant association was found between the methylation of EYA4 and differentiation, tumor size or TNM stage (Table 1).
In addition, we detected the expression of EYA4 by IHC in 30 ESCC tumor tissues and 10 non-tumor tissues and found that 63%   Similarly, wound healing assays demonstrated that ESCC cells exhibited faster rates of wound healing when EYA4 was silenced (Figure 3E).
To further elucidate the inhibitory effects of EYA4 on tumor metastasis, experimental metastasis assays were performed.
KYSE30-shEYA4 or control cells were intravenously injected into the lateral tail vein of nude mice. After 8 weeks, the mice were killed and the lungs were harvested. The number of metastatic nodules on the surface of the lungs was significantly higher in mice injected with KYSE30-shEYA4 cells than that injected with control cells (Figure 3F). H&E staining confirmed that the nodules on the surface of the lungs were metastatic tumors. Our data indicate that EYA4 is involved in the control of ESCC metastasis in vivo.
In contrast, KYSE180 and KYSE450 cells were stably transfected with the EYA4 construct, and ectopic expression of the EYA4 in these cells was determined ( Figure 4A). Transwell assay showed that EYA4 overexpression in KYSE180 and KYSE450 cells was associated with decreased migratory ability ( Figure 4B).
F I G U R E 3 Suppression of EYA4 promotes epithelial-mesenchymal transition (EMT) and invasion of human esophageal cancer cells. A, Quantitative RT-PCR and western blot analyses were used to detect the knockdown efficiency of EYA4 in KYSE30 and KYSE70 cells. To explore the effect of EYA4 on EMT, IF was used to assess the epithelial and mesenchymal markers' expression. The results showed that E-cadherin expression was obviously decreased, while the expression of vimentin and slug was increased in the EYA4knockdown group ( Figure 4C). Furthermore, the staining of slug is predominantly nuclear in EYA4 knockdown cells. qRT-PCR and western blotting also demonstrated that the expression of vimentin, slug, MMP2 and MMP13 were elevated in EYA4-knockdown cells but were reduced in EYA4-overexpression cells ( Figure 5A-C).
Taken together, these results suggest that EYA4 inhibits ESCC cell migration and invasion through the inhibition of EMT.

| EYA4 inhibits epithelial-mesenchymal transition via the PI3K/AKT/GSK3b/slug signaling pathway
To determine whether the Akt/GSK-3b/slug pathway is involved in EYA4-mediated EMT, we tested the expression of several proteins F I G U R E 4 EYA4 inhibits the migration and epithelial-mesenchymal transition (EMT) of human esophageal cancer cells. A, Quantitative RT-PCR and western blot analyses were used to detect the ectopic expression efficiency of EYA4 in KYSE180 and KYSE450 cells. B, Decreased cell migration and invasion caused by ectopic expression of EYA4 was determined byTranswell assay (*P < .05, **P < .01, independent Student t test). C, Representative IF images showing increased expression of vimentin and slug and decreased expression of E-cadherin in shEYA4-transfected KSYE30 cells compared with shScramble-transfected cells. Nuclei were counterstained with DAPI involved in this pathway. A significant increase in p-Akt, accompanied by an increase in p-GSK-3b and slug expression, was detected in EYA4-kncockdown cells ( Figure 5C). In contrast, we found a significant decrease in p-Akt and a slight decrease in slug expression in EYA4-overexpressing cells, while the change of p-GSK-3b expression was not obvious ( Figure 5C).
To confirm whether Akt/GSK-3b/slug inactiviation mediated the suppression of EYA4 on cell migration, we detected cell migration after LY294002 treatment in EYA4 knockdown cells and found that LY294002 significantly decreased KYSE30-shEYA4 cell migration ( Figure 5D). Western blotting also showed that LY294002 effectively decreased the expression of p-Akt, p-GSK-3b and slug induced by the silencing of EYA4 in KYSE30 cells ( Figure 5E).

| TGF-b1 induces an increase in DNMT and a decrease in EYA4 expression
To better characterize TGF-b1-induced EMT, the mRNA and protein expression of EYA4 and EMT-related genes were detected. We cells were more sensitive to cisplatin than the control cells (Figure 7A). Cisplatin-induced apoptosis assays showed that early apoptosis was significantly elevated in KYSE30-shEYA4 cells but was reduced in EYA4-overexpression cells ( Figure 7B). Furthermore, we assessed the expression of cH2AX and Ser-139 phosphorylation of H2AX in KYSE30-shEYA4 cells after cisplatin treatment, and found markedly higher expression of Ser-139-phosphorylated H2AX in KYSE30-shEYA4 cells in response to cisplatin ( Figure 7C).
These results suggest that EYA4 plays a role in DNA damage repair in ESCC.

| DISCUSSION
EYA4 is a key regulator of cell apoptosis and angiogenesis and has been shown to be associated with human cancers. The majority of previous studies have reported that EYA4 is frequently methylated and that it functions as a potential tumor suppressor gene in some types of cancer. In non-small-cell lung cancer, EYA4 was found to be frequently biallelically inactivated, and its low expression has been associated with poor survival of patients with sporadic lung cancers. Restoration of EYA4 expression inhibits the growth and colony formation ability of lung adenocarcinoma cells. 16 Using the Illumina Human Methylation 450K array, EYA4 was found to be methylated in more than 90% of CIMP-H colorectal cancers. 23 In addition, EYA4 functioned as a tumor suppressor gene in pancreatic ductal adenocarcinoma. 24 However, EYA4 has been reported to promote the adhesion and migration of malignant peripheral nerve sheath tumor cells and to play an oncogenic role in those tumors. 18 Thus, it seems likely that the role and function of EYA4 greatly depend on the context in which it is expressed. F I G U R E 5 EYA4 inhibits the Akt/GSK-3b/Slug pathway to inhibit epithelial-mesenchymal transition (EMT). A, Relative expressions of Ecadherin, vimentin, slug, MMP2 and MMP13 were compared by quantitative RT-PCR between (A) EYA4-knockdown and control cells and (B) EYA4-overexpression and control cells. Western blots comparing EYA4-knockdown and EYA4-overexpression cells with their respective control cells are seen in relative expression of (C) Akt, p-Akt-S473, GSK-3b, p-GSK-3b. b-actin was used as a loading control. D, The representative figures and data of Transwell assay for shEYA4-transfected KSYE30 cells and shScramble-transfected cells after PI3K inhibitor LY294002 treatment (20 lmol/L) (**P < .01, ***P < .001, independent Student t test). E, Western blot analysis of the effects of LY294002 on the E-cadherin, slug, Akt, p-Akt-S473, GSK-3b, p-GSK-3b protein levels in EYA4-knockdown cells and control cells GSK-3b signaling pathway is involved in the progression of EMT in human malignancies, such as gastric cancer, hepatocellular carcinoma and prostate cancer. [29][30][31] In this study, we found that in EYA4-knockdown cells, the expression of p-AKT, p-GSK-3b and slug were significantly increased. Moreover, an immunofluorescence assay TGF-b1 is one of the important inducers of EMT in tumor cells.
In the present study, we found that TGF-b1 could markedly induce the downregulation of EYA4 in both a dose-dependent and a timedependent manner in ESCC cells, which was accompanied by remarkable changes in morphology and expression of EMT-related molecules. In addition, TGF-b1 promoted the expression of DNMT1 and DNMT3A, especially DNMT3A, which suggests that TGF-b1 plays a certain role in the regulation of EYA4 expression in ESCC. In other human malignancies, TGF-b1 has also been found to affect transcription and expression of downstream target genes via the regulation of DNMT expression. [35][36][37] In ovarian cancer cells, TGF-b1 induced the expression of DNMT and led to extensive genomic hypermethylation, which influenced the transcription of EMT-related genes and promoted the EMT process. 38 In hepatocellular carcinoma, TGF-b1 transformed HCC cells into cells with a spindle-shaped morphology, which was accompanied by hypermethylation of CDH1 and the methylation of its promoter. 39 However, several studies have found that TGF-b1 downregulated the expression of DNMT, which resulted in the demethylation of downstream genes. 40  results in the formation of cH2AX foci, which recruits other DNA damage response proteins and DNA repair-related proteins. 41 In our study, after cisplatin-induced DNA damage in ESCC cells, we found that EYA4 promoted the repair of DNA damage, inhibited apoptosis induced by cisplatin, and maintained genomic stability. Taken together, these data suggest that EYA4 not only regulates the invasion and metastasis abilities of ESCC cells, but it also plays a role in the process of carcinogenesis through the regulation of the stability of genomic DNA.
In summary, our study reveals that EYA4 is frequently methylated in ESCC tissues and cell lines and that EYA4 inhibits cell migration and invasion through the inactivation of the Akt/GSK-3b/slug pathway and the inhibition of EMT. Moreover, EYA4 plays an important role in DNA damage repair in ESCC cells. Therefore, this protein might be a potential target for anti-metastatic drugs in future clinical practice.

CONFLI CT OF INTEREST
The authors have no conflict of interest to declare.