The Prognostic Significance of Histone Demethylase UTX in Esophageal Squamous Cell Carcinoma

The dysregulation of the ubiquitously transcribed TPR gene on the X chromosome (UTX) has been reported to be involved in the oncogenesis of several types of cancers. However, the expression and significance of UTX in esophageal squamous cell carcinoma (ESCC) remains largely undetermined. Immunohistochemistry was performed in 106 ESCC patients, and correlated with clinicopathological features and survival. The functional role of UTX in ESCC cells was determined by UTX-mediated siRNA. Univariate analyses showed that high UTX expression was associated with superior overall survival (OS, p = 0.011) and disease-free survival (DFS, p = 0.01). UTX overexpression was an independent prognosticator in multivariate analysis for OS (p = 0.013, hazard ratio = 1.996) and DFS (p = 0.009, hazard ratio = 1.972). The 5-year OS rates were 39% and 61% in patients with low expression and high expression of UTX, respectively. Inhibition of endogenous UTX in ESCC cells increased cell viability and BrdU incorporation, and decreased the expression of epithelial marker E-cadherin. Immunohistochemically, UTX expression was also positively correlated with E-cadherin expression. High UTX expression is independently associated with a better prognosis in patients with ESCC and downregulation of UTX increases ESCC cell growth and decreases E-cadherin expression. Our results suggest that UTX may be a novel therapeutic target for patients with ESCC.


Introduction
Esophageal cancer ranks as the ninth leading cause of cancer deaths in Taiwan, and more than 90% of esophageal cancer was esophageal squamous cell carcinoma (ESCC) [1]. Despite advances in the diagnosis and treatment of ESCC in recent decades, the prognosis of patients with ESCC still remains unsatisfactory [2][3][4]. The 5-year survival rate of patients diagnosed with ESCC is approximately A total of 106 patients with ESCC who had received surgery were considered in this study. The patients had a median age of 55 years (range, 29-80 years), and the characteristics of the patients are further summarized in Table 1. Among them, 101 (95%) were men and 5 (5%) were women. In terms of T classification, 42 (40%) of the patients were T1; 28 (26%) were T2; 26 (25%) were T3; and 10 (9%) were T4. Furthermore, in terms of N classification, 70 (66%) of the patients were N0; 25 (24%) were N1; 9 (8%) were N2; and 2 (2%) were N3. In terms of the 7th edition American Joint Committee on Cancer AJCC stages staging system 5 (5%) of the patients were stage IA, 17 (16%) were stage IIA; 26 (24%) were stage IIB; 11 (10%) were stage IIIA; 3 (3%) were stage IIIB; 9 (9%) were stage IIIC; and 2 (2%) were stage IV. Further analyses of histologic grades showed a grade 1 lesion in of the 10 (9%) patients, grade 2 in 70 (66%) of the patients, and grade 3 in 26 (25%) of the patients. Primary tumor location was found to be upper in 19 (18%) of the patients, middle in 36 (34%) of the patients, and lower in 51 (48%) of the patients. Among all 106 patients, resection margins were positive for residual tumor in 15 (14%) of the patients. At the time of analysis, the median periods of follow-up were 66 months (range, 61-112 months) for the 46 survivors and 53 months (range, 3.5-112 months) for all 106 patients. The 5-year survival (OS) and disease-free survival (DFS) of these 106 patients were 48% and 43%, respectively.

Correlation between Clinicopathologic Parameters and UTX Expression
Among the 106 patients considered, high UTX expression was identified in 44 (42%) of the patients (Figure 1). The associations between the clinicopathological parameters and UTX expression are summarized in Table 2. We did not observe any association between UTX expression and any clinicopathologic parameters including age, primary tumor location, histologic grading, T classification, N classification, and 7th edition American Joint Committee on Cancer (AJCC) Stage.

Correlation between Clinicopathologic Parameters and UTX Expression
Among the 106 patients considered, high UTX expression was identified in 44 (42%) of the patients (Figure 1). The associations between the clinicopathological parameters and UTX expression are summarized in Table 2. We did not observe any association between UTX expression and any clinicopathologic parameters including age, primary tumor location, histologic grading, T classification, N classification, and 7th edition American Joint Committee on Cancer (AJCC) Stage.
Moreover, we further tested whether the combination of UTX and E-cadherin expression can improve the prognostic value. We found that the combination of low UTX and low E-cadherin expression robustly identifies the group of patients with extremely poor prognosis. The 5-year OS (p = 0.001) and DFS (p < 0.001) rates were 31% and 22%, respectively, in the 36 patients with low UTX and E-cadherin expression, and 57% and 53%, respectively, in the 70 patients without low UTX and E-cadherin expression. in patients with high UTX expression, and 39% and 32% respectively, in patients with low UTX expression. Moreover, we further tested whether the combination of UTX and E-cadherin expression can improve the prognostic value. We found that the combination of low UTX and low E-cadherin expression robustly identifies the group of patients with extremely poor prognosis. The 5-year OS (p = 0.001) and DFS (p < 0.001) rates were 31% and 22%, respectively, in the 36 patients with low UTX and E-cadherin expression, and 57% and 53%, respectively, in the 70 patients without low UTX and E-cadherin expression.

Inhibition of UTX Promoted ESCC Cells Proliferation and Epithelial-Mesenchymal Transition Process
To study the functions of UTX in ESCC, we generated UTX-depleted ESCC cells in the TE8 and TE14 cell lines. The knockdown efficiency was revealed to be effective through Western blotting analysis ( Figure 3A). Next, to examine if UTX modulated ESCC proliferation, we performed MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. As shown in Figure 3B, TE8 and TE14 cells transfected with UTX-mediated siRNA were grew faster than those transfected with siControl cells. Using the same cell panels, similar results were also observed with a bromodeoxyuridine BrdU incorporation assay ( Figure 3C). Moreover, UTX-depleted TE8 and TE14 cells reduced the expressions of epithelial marker E-cadherin and increased those of Vimentin, a mesenchymal marker ( Figure 3D). Together, these results reveal that UTX prevention may induce cell proliferation and participate in the epithelial-mesenchymal transition (EMT) process in ESCC cells. To evaluate the potential relevance of the above in vitro findings in ESCC in a clinical setting, we also analyzed the protein expressions of E-cadherin in tissues samples from the 106 human ESCC patients using immunohistochemistry. There was a significant correlation between UTX expression and E-cadherin expression (p = 0.008, Table 2). Representative staining results of E-cadherin expression are shown in Figure 1.

Inhibition of UTX Promoted ESCC Cells Proliferation and Epithelial-Mesenchymal Transition Process
To study the functions of UTX in ESCC, we generated UTX-depleted ESCC cells in the TE8 and TE14 cell lines. The knockdown efficiency was revealed to be effective through Western blotting analysis ( Figure 3A). Next, to examine if UTX modulated ESCC proliferation, we performed MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. As shown in Figure 3B, TE8 and TE14 cells transfected with UTX-mediated siRNA were grew faster than those transfected with siControl cells. Using the same cell panels, similar results were also observed with a bromodeoxyuridine BrdU incorporation assay ( Figure 3C). Moreover, UTX-depleted TE8 and TE14 cells reduced the expressions of epithelial marker E-cadherin and increased those of Vimentin, a mesenchymal marker ( Figure 3D). Together, these results reveal that UTX prevention may induce cell proliferation and participate in the epithelial-mesenchymal transition (EMT) process in ESCC cells. To evaluate the potential relevance of the above in vitro findings in ESCC in a clinical setting, we also analyzed the protein expressions of E-cadherin in tissues samples from the 106 human ESCC patients using immunohistochemistry. There was a significant correlation between UTX expression and E-cadherin expression (p = 0.008, Table 2). Representative staining results of E-cadherin expression are shown in Figure 1.

Discussion
As is well known, epigenetic modifications, such as histone methylation or demethylation are involved in gene activation. UTX, a histone demethylase, removes di-and tri-methyl groups on histone H3 lysine 27 (H3K27) which is essential for tissue differentiation, cell maintenance, cell reprogramming and cancer development [20]. Using exome-or genome-wide sequencing strategies and TCGA databases, various somatic mutations and deletions of UTX have been found in several human cancers, with a prevalence of 24.24% in bladder cancer, 10% in prostate cancer, and 1.85% in esophageal cancer [20]. Interestingly, induction of UTX expression in UTX-depleted cells results in a K3K27me3 decrease and attenuates cell proliferation [11]. In addition, in normal fibroblast cells, UTX expression activates the Rb (Retinoblastoma) pathway to suppress cell growth, indicating that UTX may play the role of tumor suppressor in human cancers [21]. Conversely, in breast tumors, UTX is rarely mutated and its upregulation is correlated with poor prognosis [22]. UTX knockdown in leukemia cell lines exhibits an anti-growth effect [23]. These conflicting results imply that the function of UTX in human cancers may depend on cell-context manner. In the present study, we found that positive UTX expression was associated with better clinical outcomes in 106 patients with ESCC receiving esophagectomy and blockage of endogenous UTX by the siRNA approach led to an increase in cell proliferation and elevation of the BrdU incorporation ability in ESCC cells, suggesting that UTX acts a tumor suppressor in ESCC.
EMT plays a critical role in cancer progression. EMT allows cells to prevent death and promotes migratory and invasive abilities. An abundance of evidence indicates that an epigenetic regulator plays a vital role in controlling EMT and cancer progression. Several previous studies [24] have shown that reduced E-cadherin expression is a poor prognosticator in patients with ESCC. In our study, we found that decreased E-cadherin expression was associated with higher T classification, higher N classification, advanced 7th edition AJCC stages, and inferior OS rates in 106 ESCC patients receiving esophagectomy. With regard to colon cancer cells, Zha et al. [25] and Zhou et al. [8] reported the inactivation of UTX down-regulated E-cadherin gene expression. For breast cancer cells, Choi et al. [26] found that UTX loss induced EMT. However, the role of UTX in the regulation of EMT expression is still unclear in ESCC cells. Here, we demonstrated that loss-of function of UTX decreased E-cadherin expression and increased vimentin expression in ESCC cells, indicating that UTX might be involved in the regulation of the EMT process in ESCC.
Our study has one critical limitation in that it is a retrospective analysis and is based on a relatively small number of patients.
In conclusion, high UTX expression is independently associated with better prognosis in patients with ESCC. In ESCC cell lines, downregulation of UTX increases cell growth and decreases E-cadherin expression. Our results may further elucidate the role of UTX in ESCC and provide a potential therapeutic target for patients with ESCC.

Patient Population
We retrospectively reviewed ESCC patients receiving esophagectomy at Kaohsiung Chang Gung Memorial Hospital. Approval to analyze and publish the aggregated anonymous data was given by the Institutional Review Board committee of Kaohsiung Chang Gung Memorial Hospital at Kaohsiung, Taiwan. The approval number of this project was 104-7233B. The approval date was on 17 November 2015. We excluded patients with synchronous cancers in other organs and patients receiving preoperative chemoradiotherapy, preoperative chemotherapy, or preoperative radiotherapy. Finally, 106 patients were identified. Patients undergoing surgery had a radical esophagectomy with cervical esophagogastric anastomosis (McKeown procedure) or an Ivor Lewis esophagectomy with intrathoracic anastomosis, reconstruction of the digestive tract with a gastric tube, and pylorus drainage procedures. All patients received two-field lymph node dissection. The pathological tumor node metastasis (TNM) staging system was determined according to the 7th edition AJCC staging system [27]. The OS rate was determined from the time of surgery to death as a result of all causes. The DFS rate was computed from the time of surgery to the recurrence of disease or death from any cause without evidence of recurrence.

Immunohistochemistry
Immunohistochemistry staining was performed using an immunoperoxidase technique. Staining was performed on slides (4 mm) of formalin-fixed, paraffin-embedded tissue sections with primary antibodies against UTX (Clone D3Q1l, 1:400, Cell Signaling Technology, Boston, MA, USA) and E-cadherin (BD610182, 1:2000, BD Biosciences, Sparks, MD,). Briefly, after deparaffinization and rehydration, the retrieval of the antigen was performed by treating the slides in 10 mmol/L citrate buffer (pH 6.0) in a hot water bath (95 • C) for 20 min. Endogenous peroxidase activity was blocked for 15 min in 0.3% hydrogen peroxide. After blocking with 1% goat serum for 1 h at room temperature, the sections were incubated with primary antibodies for at least 18 h at 4 • C overnight. Immunodetection was performed using the LSAB2 kit (Dako, Carpinteria, CA, USA) followed by 3-3 -diaminobenzidine for color development and hematoxylin for counterstaining. Incubation without the primary antibody was used as a negative control, and normal colon mucosa was used as a positive control for UTX and E-cadherin. The staining was assessed by 2 pathologists (Sheng-Lan Wang and Wan-Ting Huang without any information on clinicopathologic features or treatment outcome. A semi-quantitative immunoreactive score (IRS) was used to evaluate the immunohistochemistry staining. [28] The IRS was calculated by multiplying the staining intensity (graded as: 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) with the percentage of positively stained cells (0 = no stained cell, 1 = <10% of stained cells, 2 = 10-50% of stained cells, 3 = 51-80% of stained cells, and 4 = >80% of stained cells). The criterion for positive staining was a specimen with a IRS > 4.

Cell Lines and siRNA Transfection
The human esophageal cell lines TE8 were obtained from the American Type Culture Collection, and maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum at 37 • C and 5% CO 2 . For siRNA transfection experiments, two synthetic UTX targeting siRNAs and one sicontrol were applied. Cells were transfected with UTX-mediated siRNA (50 nM) in serum-free DMEM using Plus/Lipofetamin Transfection Reagent according to the manufacturer's instructions. Two double-stranded synthetic RNA oligomers (5 -GAACAGCUCCGCGCAAAUA-3 and 5 -GAGAGUAAUUCACGAAAGA-3 ) deduced from human UTX, and one siRNA control (#4611G; Ambion, Austin, TX, USA) were used in the siRNA experiments.

Western Blotting Assay
TE8 and TE14 cells transfected with a negative control or UTX-siRNA were harvested and homogenized in RIPA (Radio-Immunoprecipitation Assay) lysis buffer, containing protease inhibitor cocktail on ice for 30 min. The homogenate was centrifuged at 4 • C at 15,000 rpm for 30 min, and lysates were collected. Protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher, Waltham, MA, USA). The cell lysates were subjected to the 10% SDS-PAGE gels for following western blotting. The primary antibodies (anti-UTX, anti-E-cadherin, anti-Vimentin, and anti-β-actin) were obtained from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Sigma (St. Louis, MO, USA).

MTT and BrdU Assay
Cells were seeded at a density of 1 × 10 4 cells per well in 96-well plates and maintained in DMEM supplemented with 10% FBS (fatal bovine serum). After overnight incubation, the culture medium was removed and the cells were washed with PBS (phosphate buffer saline). The freshly completed DMEM (Dulbecco's Modified Eagle Medium) medium was added and cultured for 48 h. Following this that the culture medium was removed and the cells were washed with PBS. The cells were then incubated with 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), in a culture medium without FBS, for 4 h at 37 • C in a 5% CO 2 atmosphere. The medium was removed and 100 µL DMSO (Dimethyl sulfoxide) buffer was added and incubated in the dark for 10 min. Absorbance was measured on a microplate reader at 540 nm. For bromodeoxyuridine (BrdU) incorporation assay, cells were seeded at a density of 5 × 10 3 cells/well in 96-well culture plates for 48 h. BrdU incorporation analysis was performed using a cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions.

Statistical Analysis
For patient data, statistical analysis was performed using the SPSS 17 software package (SPSS Inc., Chicago, IL, USA). The chi-square test and Fisher's exact test were used to compare data between the two groups. For the survival analysis, the Kaplan-Meier method was used for univariate analysis, and the difference between the survival curves was tested by a log-rank test. In a stepwise forward fashion, significant parameters at a univariate level were entered into a Cox regression model to analyze their relative prognostic value. For all analyses, two-sided tests of significance were used with p < 0.05 being considered significant.