Overexpressed HDAC4 is associated with poor survival and promotes tumor progression in esophageal carcinoma

HDAC4 is upregulated in ESCC tissues and EC cell lines

The mRNA expression of HDAC4 was analyzed in 86 paired ESCC and adjacent normal tissues by qRT-PCR, which was normalized using GAPDH as an internal control. The results showed that HDAC4 expression was significantly up-regulated in tumor tissues compared with the paired normal tissues (P<0.001) (Fig. 1A). HDAC4 mRNA was also determined in cell lines, which shows significantly higher expression in EC/CUHK1, KYSE30, KYSE140, KYSE150, and KYSE180 EC cell lines than the immortalized human esophageal cell line NE1 (P< 0.05) (Fig. 1B). Western blot indicates that HDAC4 protein is also significantly higher in 8 ESCC tissues than the matched adjacent normal tissues (P< 0.05) (Fig. 1C-D), further confirming that HDAC is overexpressed in ESCC.

Figure 1. The expression of HDAC4 is upregulated in ESCC tissues and cell lines and associated with survival. (A) Real-time qRT-PCR analysis shows that expression of HDAC4 mRNA is significantly increased in 86 ESCC tissues compared with the corresponding normal tissues. **** P <0.001; (B) The expression of HDAC4 mRNA is significantly increased in EC cell lines, EC/CUHK1, KYSE30, KYSE140, KYSE150 and KYSE180 cell lines, compared with the human normal esophageal cell line NE1. The expression was normalized by GAPDH mRNA. * P <0.05; (C) Western blot analysis of HDAC4 protein expression in eight ESCC tissues and paired normal tissues; GAPDH was used as a loading control. (D) HDAC4 protein expression in eight ESCC tissues and paired adjacent tissues was quantitated using ImageJ software (Wayne Rashband). (E) Kaplan–Meier analysis shows that HDAC4 overexpression is significantly associated with poor overall survival in 86 ESCC cases (P <0.001, log-rank test). (F) Kaplan–Meier analysis shows that expression of HDAC4 is marginally significantly associated with poor relapse-free survival in 86 ESCC cases (P =0.056, log-rank test).

High HDAC4 expression is associated with unfavorable clinical characteristics in ESCC patients

To investigate the clinical significance of HDAC4 overexpression, we analyzed the relationship between HDAC4 mRNA expression and clinical characteristics in ESCC patients. ESCC patients were divided into high and low HDAC4 expression groups using the median expression level as the cut-off point (median, 2.48; range, 0.30 - 6.90). The association between HDAC4 expression level and clinical characteristics was evaluated by Chi-square test. The results show that high HDAC4 expression is significantly correlated with high pathological grade (poor differentiation), T stage, N stage and advanced TNM stage in ESCC patients (Table 1), indicating that HDAC4 overexpression is associated with ESCC progression.

HDAC4 is associated with poor prognosis and an independent prognostic factor in ESCC patients

To further explore the prognostic value of HDAC4 in ESCC patients, we analyzed overall survival (OS) and progression-free survival (PFS) of ESCC patients with high or low HDAC4 expression using Kaplan–Meier method and Log-rank test. The 5-year OS and PFS of patients with high HDAC4 expression were only 20.15% and 30.92%, which were considerably shorter than those with HDAC4 low expression (68.75% and 53.59%) (Fig. 1E-1F). Thus, high level of HDAC4 is significantly correlated with poor prognosis of ESCC patients.

To determine whether HDAC4 is an independent prognostic factor in ESCC patients, we analyzed the relationship of HDAC4 expression and various clinicopathological parameters with patient survival using univariate and multivariate Cox proportional hazard models. Univariate analysis indicated that N stage, TNM stage and HDAC4 expression are significant predictors for OS of ESCC patients (Table 2). Moreover, N stage and TNM stage are also significant predictors for PFS in ESCC patients, and HDAC4 is only a marginally significant predictor for PFS in ESCC patients. Multivariate Cox regression analysis demonstrated that HDAC4 is an independent risk predictor for OS (HR: 2.846, 95% CI: 1.456-5.566, P = 0.002) in ESCC patients, not for PFS (Table 2). These results suggest that HDAC4 is a potential biomarker for predicting the overall survival of ESCC patients.

We further stratified ESCC patients by different TNM stage. The results show that stage I-II patients with high level of HDAC4 mRNA have remarkably poorer OS than those with low level (Supplementary Fig. 1A left panel), and stage III patients with high HDAC4 level have marginally significantly shorter OS than those with low level (Supplementary Fig. 1B left panel). It should be noted that the latter is likely to be due to the small sample size. No significant difference is seen between PFS of patients with high and low HDAC4 expression in both stage I-II and stage III subgroups.

Therefore, HDAC4 predicts the survival of ESCC patients independently of clinical stage, particularly in OS, indicating that HDAC4 can provide additional prognostic information for the clinical staging system.

HDAC4 is important for EC cell proliferation

To assess the biological significance of HDAC4 in EC cells, we transfected EC/CUHK1 and KYSE30 cells with two HDAC4 siRNAs and one control (NC) siRNA. The efficiency of siRNA mediated HDAC4 knockdown was evaluated by qRT-PCR and western blot, which show significant reduction of both HDAC4 mRNA and protein (Fig. 2A-2B for mRNA, Fig. 3E and Fig. 4D for protein). The CCK8 cell proliferation assay was further performed. The result shows that HDAC4 siRNAs, not NC siRNA, significantly reduce proliferation of KYSE30 and EC/CUHK1 cells (Fig. 2C and 2D). These results indicate that HDAC4 is important for EC cell proliferation.

Figure 2. HDAC4 silencing inhibits cell proliferation. Real-time qRT-PCR analysis of HDAC4 mRNA expression in (A) KYSE30 and (B) EC/CUHK1 cell lines transfected with HDAC4 specific siRNAs or a control siRNA. CCK8 assay was then performed to evaluate cell growth of (C) KYSE30 and (D) EC/CUHK1 cells). Each value represents the mean ± http://S.D.in three independent experiments. * P<0.05; ** P< 0.01. OD, optical density.

Figure 3. HDAC4 knockdown induces G1/S arrest in EC cells. (A and C) Flow cytometry analysis of indicated EC cells transfected with HDAC4-specific siRNAs or a control siRNA; G1-phase cells are significantly increased while S-phase cells are reduced in HDAC4 knockdown (B) KYSE30 and (D) EC/CUHK1 cells, * P <0.05; (E) HDAC4 down-regulation increases the expression of p21 and p27 proteins while reduces pRb, CDK2 and CDK4 in EC cells.

Figure 4. Down-regulation of HDAC4 inhibits the migration of EC cells. (A) Knockdown of HDAC4 reduces migration of KYSE30 and EC/CUHK1 cells. EC cells were transiently transfected with HDAC4 siRNA or a control siRNA. The number of migrated cells is decreased in cells (x100 magnification) transfected with HDAC4 siRNAs compared with those transfected with a control siRNA. Wound healing assay demonstrates that down-regulation of HDAC4 inhibits cell migration at 12 h and 24 h in (B) KYSE30 and (C) EC/CUHK1 cells (magnification, x100). (D) Western blot analysis indicates that knockdown of HDAC4 increases the expression of E-cadherin and α-catenin, and decreases the expression of Vimentin.

HDAC4 promotes G1/S cell cycle progression in EC cells through downregulating p21/p27 and upregulating CDK2/4 and Rb phosphorylation

Because HDAC4 knockdown inhibits proliferation of EC cells, we asked whether cell cycle is affected by HDAC4 knockdown. Flow cytometry analysis shows that HDAC4 downregulation significantly increases G1 cell population while decreases S cell population in KYSE30 cells (Fig. 3A-B). A similar result is obtained in EC/CUHK1 cells (Fig. 3C-D). These results indicate that HDAC4 knockdown causes G1 cell cycle arrest in both EC cell lines.

The retinoblastoma (Rb) protein is important for G1/S cell cycle transition, which is regulated by two cyclin-dependent kinase (CDK) complexes, CDK4/6-Cyclin D and CDK2-Cyclin E. The CDK inhibitors p21 and p27 oppose the effect of CDK4/6-Cyclin D and CDK2-Cyclin E. We examined whether HDAC4 knockdown affects the level of Rb phosphorylation (pRb), p21/p27 and CDK2/4. Indeed, HDAC4 knockdown in EC/CUHK1 and KYSE30 cells causes a decrease in CDK2/4 and pRb, but an increase in p21 and p27 (Fig.3E). Taken together, our results demonstrate that HDAC4 is important for EC cell cycle progression through CDK2/4, pRb and p21/p27.

HDAC4 promotes migration of EC cells through enhancing epithelial-mesenchymal transition (EMT)

We also investigated the potential role of HDAC4 in EC cell migration using transwell and wound healing assays. In transwell assay, down-regulation of HDAC4 significantly reduces the number of migrated cell in KYSE30 and EC/CUHK1 cells by approximately 60 % and 80%, respectively (Fig. 4A). In wound healing assay, cell migration rate is markedly decreased in HDAC4 knock-down cells compared with control cells (by approximately 65% and 50% at 12 h and 24 h, respectively) (Fig. 4B-C).

Because HDAC4 knock down inhibits migration of EC cells, we asked if EMT is involved. To this end, Western blot analysis was carried out to determine the expression of E-Cadherin, α-catenin and Vimentin markers for MET. The expression of the epithelial markers E-cadherin and α-catenin are increased, whereas that of the mesenchymal marker Vimentin is reduced in EC/CUHK1 and KYSE30 cells following HDAC4 knockdown (Fig. 4D). These results demonstrate that HDAC4 down-regulation inhibits EMT.

Tumor samples and cell lines

A total of 28 primary ESCC patients who underwent curative esophagectomy between October 2008 and September 2009 at Cancer Center of Guangzhou Medical University (Guangzhou, Guangdong, China) were enrolled in this study. Another cohort of 58 ESCC patients was recruited at Sun Yat-Sen University Cancer Center, Guangzhou, in 2009. ESCC specimens and paired adjacent normal esophageal epithelium tissues from all 86 patients were collected and snap frozen in liquid nitrogen and stored at −80°C until further use for qRT-PCR and western blot assay. None of the patients received radiotherapy or chemotherapy before surgery. Clinical and pathological information is listed in Table 1. This study was approved by the Research Ethics Committees of Cancer Center of Guangzhou Medical University and Sun Yat-Sen University Cancer Center, and written informed consent was obtained from all patients.

Human cell lines EC/CUHK1, an esophageal carcinoma cell line, and KYSE30, KYSE140, KYSE150, and KYSE180, which are esophageal squamous cell carcinoma cell lines, were cultured at 37°C and 5% CO₂ in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Inc.) with 10% Gibco fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). NE1 cell line, a normal esophageal cell line, was maintained in a 1:1 mixture of defined keratinocyte serum free medium with growth supplements (Invitrogen) and EpiLife medium with 60 uM Calcium (Invitrogen).

Total RNA extraction

The total RNA was extracted from tissues and cultured cells using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. The quality and concentration of RNA were determined using a NanoDrop® ND-2000 spectrophotometer (Thermo Fisher Scientific, Inc.).

Real-time qRT-PCR analysis

Total RNA (400 ng) was employed as template for the reverse transcription (RT) reaction using GoScript Reverse Transcription (RT) System (Promega, USA) according to manufacturer's instructions. Real-time qPCR was performed with 1 μL of RT products (cDNA) and 5μL of GoTaq qPCR Master Mix (Promega, USA) in a 10-μL total volume on a Light Cycler 480 instrument (Roche, USA) according to the manufacturer's protocol. Each sample was analyzed in triplicate wells, and the reactions without cDNA also were included as negative control. The conditions of thermal cycling were as follows: 95°C at 10 min for a hot start, then 45 cycles at 95°C for 15 s, 60°C for 60 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. The primers were as listed below: HDAC4, forward 5′-GAG AGA CTC ACC CTT CCC G-3′ and reverse 5′-CCG GTCTGC ACC AACCAA G-3′; GAPDH, forward 5′-CTCCTCCT GTTCGACAGTCAGC-3′and reverse 5′-CCCAATAC GACCAAATCCGTT-3′. The 2-^ΔΔCq equation was used to represent the relative expression of HDAC4 and Student's t-test was used to analyze the results.

siRNA transfection

The HDAC4 siRNA1, siRNA2 and negative control (NC) were designed and synthesized by Shanghai GenePharma, Ltd. (Shanghai, China). When the cells reached 60% confluence, 100 nM HDAC4 siRNA1, siRNA2 and NC were transfected into the cells with Invitrogen Lipofectamine® 2000 RNAiMAX reagent (Thermo Fisher Scientific, Inc.), respectively, according to the manufacturer's protocol.

Cell viability assays (CCK8)

Following transfection for 36 h, EC cells were plated and cultured in 96-well plates (1.0×10³ cells/well, six wells per group) for an additional 4 days. The viability of the cells was measured using CCK8 Cell Counting Kit (JingXin Biological Technology, Guangzhou, China). At a specific time every day for 4 days, 10 μl CCK8 was added to each well, followed by additional incubation for 2 h at 37°C. The cells in the 96-well plate were gently agitated for 10 min and the absorbance was measured at 450 nm (SpectraMax® M5 Multi-Mode Microplate Reader; Molecular Devices LLC, Sunnyvale, CA, USA) to calculate the cell viability.

Cell cycle analysis

EC cells were trypsinized, washed in ice-cold PBS, and fixed in 75% ice-cold ethanol. Cell cycle analysis was conducted by flow cytometry. Briefly, the fixed cells were centrifuged at 1000 r.p.m. and washed twice with ice-cold PBS. Propidium iodide (BD Biosciences, Lexington, KY, USA) staining solution (50 mg/ml, final concentration) were then added and the cells were incubated at 37°C for 30 min in the dark. Cell cycle analysis (20,000 cells per sample) was carried out in a flow cytometer (ACEA NovoCyte^TM, San Diego, California, USA). Cell cycle analysis software (NovoExpress^TM, San Diego, California, USA) was used to determine the percentage of cells at each phase of the cell cycle.

Transwell assay

A Boyden chamber Transwell assay (24-well Transwell plate; BD Biosciences, Franklin Lakes, NJ, USA) was used to measure cell migration, and was performed according to the manufacturer's protocol. EC cells were trypsinized, washed three times with PBS, resuspended (1×10⁵ cells/150 μl) in Serum-free medium, and added to the upper chamber, while 500 μl 10% FBS-containing medium was added to the lower chambers. Following incubation for 24 h, non-migrating cells were removed by a cotton swab, and migrated cells in the filters were immersed in 100% methanol for 15 min at room temperature, and then stained with 0.1% crystal violet (Weijia Biology Science and Technology Co., Ltd) for 30 min at room temperature prior to washing with water. The migrated cells were counted for five random fields with an inverted microscope (BX53; Olympus Corp., Tokyo, Japan). The experiments were conducted in triplicate and repeated three times.

Wound healing assay

Forty-eight hours after siRNA transfection, a linear scratch wound was performed using a plastic micropipette tip in a confluent monolayer of cells in 6-well plates. Medium without FBS was used in order to inhibit cell proliferation. Then, the cells were washed using PBS in order to wipe off the trashy cells. The remnant EC cells were maintained in RPMI-1640 medium. The distances that the cells moved were observed and recorded.

Western blotting

EC cells were lysed using the Total Protein Extraction Kit (KeyGEN BioTECH, Jiangsu, China). Following centrifugation at 14,000 x g for 10 min at 4°C, the proteins in the supernatants were quantified using a Bicinchoninic Acid Protein Assay kit (Pierce™; Thermo Fisher Scientific, Inc.) and separated using 10% SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont, UK). The membranes were blocked with 5% nonfat milk in TBST for 2h at room temperature (RT), and incubated with specific primary antibodies at 4°C for 12h, and then with horseradish peroxidase (HRP)-conjugated antibodies for 2h at RT. Detection was performed with electrochemiluminesce (ECL) (Tanon, Shanghai, China). Anti-HDAC4 (Abcam, cat. no. ab12172; 1:1000), Anti-pRb (phospho S780) (ab173289), Anti-Rb (ab85607), anti-p21 (CST, cat. no. mAb #2947; 1:1000), anti-p27 (CST, cat. no. mAb #3686; 1:1000), anti-CDK2 (CST, cat. no. mAb #2546; 1:1000), anti-CDK4 (CST, cat. no. mAb #12790; 1:1000), anti-GAPDH (CST, cat. no. mAb#5174; 1:1000), anti-E-Cadherin (Abcam, cat. no. ab 15148; 1:500), anti-α-catenin (Abcam, cat. no. ab51032; 1:5000), anti-Vimentin (CST, cat. no. mAb #5741; 1:1000), anti-β-tubulin (CST, cat. no. mAb #2148; 1:1000).

Statistical analysis

Statistical analysis was performed using Student's t test which was used to test the differences between two groups. P value of <0.05 was considered as a statistically significant difference. The association between HDAC4 and clinical characteristics of the patients was analyzed using Student's t-test, χ² test or Fisher's exact test. SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for all analysis. P<0.05 was considered to indicate a statistically significant difference.