EYA4 serves as a prognostic biomarker in hepatocellular carcinoma and suppresses tumour angiogenesis and metastasis

Abstract Eye absent homolog 4 (EYA4) has been demonstrated to be down‐regulated in hepatocellular carcinoma (HCC), but its biological function and the mechanism in HCC angiogenesis and metastasis remain largely unknown. Herein, we showed that EYA4 expression was frequently low in HCC tissue samples compared with matched adjacent non‐tumourous tissues. In the analysis of 302 HCC specimens, we revealed that decreased expression of EYA4 correlated with tumour differentiation status. Univariate and multivariate analyses identified EYA4 as an independent risk factor for recurrence‐free survival (RFS) and overall survival (OS) among the 302 patients. Functional assays showed that forced expression of EYA4 suppressed HCC cell migration, invasion and capillary tube formation of endothelial cells in vitro, as well as in vivo tumour angiogenesis and metastasis in a mouse model. Furthermore, mechanism study exhibited that EYA4 could inhibit HCC angiogenesis and metastasis by inhibiting c‐JUN/VEGFA pathway. Together, we provide proof that EYA4 is a novel tumour suppressor in HCC and a new prognostic biomarker and therapeutic target in HCC.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is the fifth most fatal cancer and the third most common cause of cancer-related deaths in the world, and the number of newly diagnosed cases continues to grow. 1 Prognosis among patients with HCC remains poor after chemotherapy or pharmacotherapy on account of high rates of recurrence and metastasis. 2 Hence, it is imperative to further investigate the molecular mechanisms underlying the progression and recurrence of HCC and to ascertain potential therapeutic targets to improve HCC treatment.
The Eyes Absent (EYA) family of proteins represents members of a highly conserved regulatory network dealing with cell fate determination of organisms from insects to humans. The EYA family contains four members-EYA1, EYA2, EYA3 and EYA4-which are the novel vertebrate genes related to the eyes absent gene in Drosophila. 3 EYA proteins are dual-role phosphatases whose threonine phosphatase and tyrosine phosphatase activities are carried out by different domains. 4 It is generally recognized that EYAs work as transcriptional coactivators recruited by the SIX protein, 5,6 and transactivation of the SIX-EYA complex depends on the tyrosine phosphatase activity of EYA. 7 As mentioned above, EYA4 belongs to the EYA family and is reported to be dysregulated in some human cancers. 8 EYA4 is inactivated in non-small cell lung cancer, and blocked EYA4 expression is associated with poor prognosis in sporadic lung cancers. 9 In addition, EYA4 is a promising tumour suppressor gene because it controls the up-regulation of DKK1 and blocks the Wnt signalling pathway in colorectal cancer. 10 Likewise, lower levels of EYA4 expression are observed in HCC tissues and are an independent predictor of both shorter disease-free survival (DFS) and overall survival (OS). 11 And more recently, EYA4 was shown to inhibit HCC cell growth and invasion by suppressing NF-κB-dependent RAP1 transactivation. 12 Nevertheless, the precise role that EYA4 plays in HCC angiogenesis and metastasis remain largely unknown.
Herein, we report that low expression of EYA4 is closely related to tumour differentiation status and poor prognosis of HCC. Our experiments showed that forced expression of EYA4 suppressed HCC angiogenesis and metastasis via inhibiting c-JUN/VEGFA pathway. These results provide not only a clearer understanding of the involvement of EYA4 in HCC progression but also a potential therapeutic target in HCC.

| Immunohistochemical staining
This staining was performed as described previously. 13 After fixing with formalin and embedding in paraffin, tissue sections were prepared, deparaffinized and rehydrated and then subjected to antigen retrieval with citrate buffer (pH 6.0). The tissue slices were incubated in 0.3% H 2 O 2 and blocked with 1% BSA for another 30 min. Next, the slices were incubated with primary antibodies at 4°C for 12 h. After that, the slices were probed with a secondary antibody at room temperature for 30 min. The Dako ChemMate TM Envision TM Detetcion Kit (DaKo, Glostrup, Denmark) was subsequently applied to detect the primary antibodies. The tissue slides were lightly counterstained with haematoxylin and photographed using an Olympus microscope (Model BX40F4, Tokyo, Japan). Integrated optical density (IOD) was measured by means of Image-Pro Plus 6.0 (IPP) and EYA4 down-regulation was defined as IOD weaker in HCC than in a paired non-tumourous tissue sample.

| Cell lines and cell culture
Human HCC cell lines, SMMC-7721 and HCC-LM3, were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All the cell lines were cultured in the DMEM medium (Gibco BRL, Rockville, MD) supplemented with 10% of fetal bovine serum (Sigma, St. Louis, MO), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA). Each cell line was cultivated at 37°C and 5% CO 2 in a humidified incubator.

| Lentiviral transduction
The lentivirus overexpressing EYA4 was purchased from Hanbio (Shanghai, China). The corresponding empty lentiviral vector served as a negative control. The EYA4 knockdown lentivirus was also obtained from Hanbio. SMMC-7721 and HCC-LM3 cells were transduced with the recombinant lentivirus in the presence of 10 μg/mL Polybrene. Forty-eight hours later, Western blot analysis was carried out for determining knockdown efficiency.

| Western blot analysis
This analysis was conducted as previously described. 14 Cells were lysed in RIPA buffer to obtain total protein samples and the proteins were

| Wound-healing assay
This assay was performed to assess the cell migration ability.
SMMC-7721 and HCC-LM3 cells with or without ectopic expression of EYA4 seeded in 6-well plates were cultured to 80%-90% confluence and then pretreated with 10 μg/mL mitomycin C (Sigma) for

| Transwell migration and invasion assays
SMMC-7721 and HCC-LM3 cells with or without ectopic expression of EYA4 were seeded onto the membrane of the upper chamber with Matrigel in a serum-free medium. The lower chamber was filled with the complete DMEM medium (containing 10% of fetal bovine serum as a chemoattractant). After incubation at 37°C for 12 h, the invading cells that got attached to the lower surface of the membrane were stained with crystal violet and counted under a microscope. The migration ability of HUVEC cells was tested using a BD Transwell assay insert with a non-coated membrane.

| Tube formation assay
HUVECs (1 × 10 4 cells/well) were pre-cultured in medium without serum for 6 h and seeded over the Matrigel-coated 96-well plate. After attached, the cells were cultured in the indicated conditioned media of SMMC-7721 and HCC-LM3 cell supernatants for 6 h. The total tube area was quantified as mean relative tube length obtained from image analysis of five random microscopic fields using Image J software.

| In vivo assays
The animal experiment was approved by the Scientific Investigation

| Statistical analysis
Data were expressed as mean ± standard deviation. Statistical analyses were conducted on a personal computer in the SPSS 23.0 statistical software (SPSS Inc, Chicago, IL). Differences between two groups were assessed by unpaired Student's t test and qualitative variables were analysed by the χ 2 test. Kaplan-Meier analyses with the log rank test were performed to evaluate recurrence-free survival (RFS) and OS. A Cox proportional hazards model was employed to analyse the independent factors affecting RFS and OS. Data with P < 0.05 were considered significant.

| EYA4 is repressed in HCC and serves as a prognostic factor in patients with HCC
To analyse the expression of EYA4 in HCC, mRNA levels of EYA4 were detected in 10 HCC tissue samples and adjacent normal tissue samples by quantitative reverse-transcription PCR (qRT-PCR).
Compared with the matched normal tissues, HCC tissue samples showed down-regulation of EYA4 mRNA ( Figure 1A). We then performed IHC analysis on the TMA that contained 302 HCC samples to assess the clinical significance of EYA4. The expression was quantified in the Image-Pro Plus 6.0 software (IPP). In agreement with the qRT-PCR results, TMA analysis showed a significantly F I G U R E 1 Eye absent homolog 4 (EYA4) is repressed in hepatocellular carcinoma (HCC) and serves as a prognostic factor among patients with HCC. (A) mRNA expression of EYA4 was determined by qRT-PCR in HCC tissue samples and adjacent normal tissues. β-actin was used as an internal control. (B) Representative immunohistochemical staining of EYA4 in the tissue microarray (TMA). (C) Normalized integrated optical density of EYA4 in HCC samples and paired non-tumourous tissue samples in TMA analysis. The low-EYA4-expression group had worse recurrence-free survival (D) and overall survival (E) as compared to the high-EYA4-expression group. The prognostic significance was evaluated by Kaplan-Meier survival analysis and the log rank test. **P < 0.01 decreased intensity of EYA4 staining in HCC tissue samples than normal tissues ( Figure 1B,C). Besides, EYA4 down-regulation was significantly associated with tumour differentiation status but not other clinical parameters (Table 1). Furthermore, we sought to determine whether down-regulation of EYA4 was associated with the prognosis of patients with HCC after hepatectomy. The log rank test revealed that patients with low EYA4 expression had unsatisfactory RFS and OS ( Figure 1D,E). The univariate analysis indicated that HBs and HBe antigens, tumour size, tumour number, microvascular invasion and EYA4 expression remarkably correlated with RFS. Additionally, these parameters correlated significantly with OS (Table 2). Our multivariate analysis revealed that low EYA4 expression was an independent risk factor for RFS and OS among patients with HCC after curative hepatectomy (Table 3).

| EYA4 inhibits HCC cell migration, invasion and angiogenesis in vitro
To evaluate the possible function of EYA4 in HCC cells, we introduced the EYA4-overexpressing lentivirus into SMMC-7721 and HCC-LM3 cells. The efficiency of the lentiviral transduction was verified by Western blotting (Figure 2A). Wound-healing and Transwell invasion assays were first conducted to explore the function of EYA4 in HCC cells. As shown in Figure 2B,C, 48 h after administration of a scratch, the gap between cells was broader in SMMC-7721 and HCC-LM3 cell groups overexpressing EYA4 than that in the negative control group, indicating that overexpression of EYA4 could reduce cell migration. In additon, the results of the Transwell invasion assay revealed that ectopic expression of EYA4 weakened the invasion ability of HCC cells ( Figure 2D).
Subsequently, we examined the effects of EYA4 overexpression on the angiogenesis using an in vitro HUVEC model. The conditioned media from EYA4-overexpressing SMMC-7721 and HCC-LM3 cells significantly attenuated the migration and tube-like structure formation of HUVECs ( Figure 2E-G), suggesting that EYA4 inhibits HCC angiogenesis in vitro.

| EYA4 regulates HCC cell invasion and angiogenesis through the c-JUN/VEGFA pathway
Next, we studied the underlying mechanism via which EYA4 suppresses HCC cell invasiveness and angiogenesis. Given that VEGFA was crucial for cancer growth and neovascularization, 15 we examined the expression of VEGFA in SMMC-7721 and HCC-LM3 cells overexpressing EYA4 or vector. We observed that overexpression of EYA4 decreased the levels of VEGFA in these cells ( Figure 3A Figure 3B). Furthermore, inhibition of c-JUN can block EYA4 knockdown-mediated promotion of HCC cell invasion ( Figure 3C). We also found that after c-JUN inhibition, conditioned media from EYA4 knockdown-transfected HCC cell cultures could significantly decrease the migration and capillary tube formation of HUVECs ( Figure 3D,E). after four weeks ( Figure 4A). As shown in Figure 4B,

| D ISCUSS I ON
In this study, we revealed that EYA4 was significantly down-regu-   progression and prognosis. 19 We found that EYA4 overexpression interfered with the pro-angiogenic activity of HCC cells, as determined by in vitro endothelial cell tube formation assay. Additionally, VEGFA expression was suppressed by EYA4, which provides an explanation for the decreased angiogenic activity in HCC cells. Hence, our data support the tumour suppressor function of EYA4 in HCC.
Although EYA4 was found to be involved in the regulation of tumour progression in HCC, the underlying molecular mechanism remains poorly understood. Previously Kim and colleagues reported that EYA4 acts as a new tumour suppressor gene in colorectal cancer and its expression was down-regulated in HCT15 colorectal cancer cells overexpressing EYA4. 12 As a transcription factor, c-JUN functions as an upstream regulator of many genes, including VEGFA and participates in tumour growth and metastasis. 20,21 Since EYA4 possessed the N-terminal serine/threonine-specific phosphatase activity, 7 it was hypothesized that EYA4 down-regulated c-JUN via dephosphorylation.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
FMG and WPZ were involved in the study conception and design.