Nuclear expression of Twist promotes lymphatic metastasis in esophageal squamous cell carcinoma

Twist-1 protein (also called Twist) has been suggested to be involved in tumor epithelial-mesenchymal transition (EMT) related progression, however, the mechanism by which twist promotes lymph node metastasis is not fully understood. In the present study, we found that nuclear twist expression is clearly correlated with lymph node (LN) metastasis as determined by immunohistochemistry (IHC). A highly invasive EC109 cell subline, EC109-P, was established by repeated in vitro transwell isolations for the cell model. Immunofluorescence (IF) assay demonstrated that nuclear twist expression was markedly higher in the highly invasive EC109-P cell line when compared with EC109 and EC9706 cells. Based on our cell model, the function and mechanism by which twist regulates LN metastasis in ESCC was investigated. The results showed that the overexpression of Twist could significantly increase the invasion and VEGF-C expression of EC9706 cells, whereas the knockdown of twist expression results in the opposite effects. This finding was further strengthened by the results of the analysis of co-expression of twist and VEGF-C by IHC in ESCC clinical samples. In summary, our study indicates that nuclear twist plays an important role in ESCC lymphatic metastasis by increasing the expression of VEGF-C. The combination of twist and VEGF-C detection could be a reliable prediction of LN metastasis in ESCC.


Introduction
Esophageal squamous cell cancer (ESCC) is one of the most common malignancy tumors, which morbidity ranks sixth among the most common types of cancers and is currently the fifth leading cause of death worldwide. 1 Although surgical advances, such as excision with clear margins, have been achieved in ESCC treatment, the overall 5-year ESCC survival rate is still unsatisfactory. 2 As it is well known that lymph node (LN) metastasis is a key prognostic factor for long-term ESCC outcomes, 3,4 an understanding of the mechanisms is critical for the prediction, prevention and cure of ESCC LN metastasis.
Twist is a transcription factor that promotes the EMT-associated with tumor metastases, cancer stem cells and drug resistance [5][6][7] and acts cooperatively with Bmi1 to inhibit the expression of the epithelial marker E-cadherin, and leads to a poor prognosis in a variety of head and neck cancers. 8 Twist can also upregulate AKT2 to enhance tumor migration and resistance to paclitaxel, 9 and increasing Twist expression was found to be correlated with lymph node metastases in colon cancer and ESCC. [10][11][12] Nevertheless, the mechanism by which Twist regulates lymph node metastasis in Twist-1 protein (also called Twist) has been suggested to be involved in tumor epithelial-mesenchymal transition (EMT) related progression, however, the mechanism by which Twist promotes lymph node metastasis is not fully understood. In the present study, we found that nuclear Twist expression is clearly correlated with lymph node (LN) metastasis as determined by immunohistochemistry (IHC). A highly invasive EC109 cell subline, EC109-P, was established by repeated in vitro transwell isolations for the cell model. Immunofluorescence (IF) assay demonstrated that nuclear Twist expression was markedly higher in the highly invasive EC109-P cell line when compared with EC109 and EC9706 cells. Based on our cell model, the function and mechanism by which Twist regulates LN metastasis in ESCC was investigated. The results showed that the overexpression of Twist could significantly increase the invasion and VEGF-C expression of EC9706 cells, whereas the knockdown of Twist expression results in the opposite effects. This finding was further strengthened by the results of the analysis of co-expression of Twist and VEGF-C by IHC in ESCC clinical samples. In summary, our study indicates that nuclear Twist plays an important role in ESCC lymphatic metastasis by increasing the expression of VEGF-C. The combination of Twist and VEGF-C detection could be a reliable prediction of LN metastasis in ESCC.

Nuclear expression of Twist promotes lymphatic metastasis in esophageal squamous cell carcinoma
ESCC is unclear. VEGF-C, a member of the vascular endothelial growth factor (VEGF) family, can lead to lymphatic vascular endothelium growth and induce selective hyperplasia of the lymphatic vasculature in vivo. 13,14 VEGF-C plays significant roles in cancer cell mobility and invasiveness by binding to its receptors. 15,16 Therefore, VEGF-C promotes lymphangiogenesis and, in turn, increases lymphatic metastasis in different cancer types including ESCC. [17][18][19][20][21][22][23] Although the level of Twist was positively correlated with VEGF-C in supraglottic carcinoma, 24 the mechanisms by which Twist and VEGF-C are involved in lymphatic metastasis in ESCC require to be further study.
To investigate the roles of the subcellular localization of Twist in ESCC progression, especially in lymphatic metastasis, in the present study, IHC was used to detect the expression of Twist in 168 ESCC tissues. We established a highly invasive cell subline, EC109-P using repetitive transwell assays. A Twist overexpression and siRNA plasmids were transfected into ESCC cell lines, and transwell assays were used to detect the alteration of invasion ability induced by the up and downregulation of Twist. Moreover, the co-expression of Twist and VEGF-C in cell lines and clinical samples were evaluated by WB, IHC and real-time PCR.
showed that the enhanced Twist expression notably increased the invasion ability of EC9706 ( Fig. 4C and D). These data are consistent with the observation that Twist promotes ESCC lymph node metastasis in tissues.
Twist promotes ESCC lymph node metastasis through increasing VEGF-C. The present study confirmed that Twist was critical in LN metastasis of ESCC. Therefore, we further investigated the mechanism of Twist in the invasion of ESCC by determining whether Twist promotes ESCC invasion through regulating VEGF-C. WB and real time PCR showed that the overexpression of Twist markedly upregulated the mRNA and protein expression of VEGFC in EC9706 cells ( Fig. 5A and C). Conversely, the knockdown endogenous Twist expression in the EC109-P cells markedly inhibited the expression of VEGF-C protein and mRNA ( Fig. 5B and D). These data indicated that Twist promoted ESCC invasion partly through increasing VEGF-C expression.
Twist expression in the nucleus is associated with VEGF-C in ESCC tissues. To determine whether the correlation of Twist and VEGF-C levels was associated with the development of lymph node metastasis in ESCC, we further examined VEGF-C in serial sections of ESSC tissues from 168 patients by IHC. The results showed no statistical correlation between Twist expression in the cytoplasm and VEGF-C expression (r = 0.121, p = 0.118, Spearman's rank test), but a significant correlation was found between nuclear Twist and VEGF-C expression ( Table 2 and Fig. 5E) (r = 0.356, p < 0.001, Spearmans rank test). Moreover,

Results
Nuclear expression of Twist in ESCC tissues associated with LN metastasis. IHC was performed to detect the expression of Twist in 168 ESCC tissues. The results showed that Twist was expressed in the nucleus and cytoplasm in ESCC tissues (Fig. 1A). Thus, we analyzed the Twist subcellular localization, as correlated with the clinical significance. The nuclear localization of Twist presented in 60.7% (102/168) of the ESCC tissues, and the positive rate in the cytoplasm was 35.1% (59/168). The Twist expression in the nucleus and cytoplasm associated with clinicopathological characteristics of the patients, including gender, age, location, T stage and LN metastasis, are presented in Table 1.
Positive expression of Twist in the nucleus but not in the cytoplasm was significantly correlated with LN metastasis (p = 0.001). In contrast, the Twist expression in both the nucleus and cytoplasm had no statistical correlation with age, sex, T stage and location (p > 0.05), indicating that Twist expression in the nucleus was critical for lymph node metastasis of ESCC.
We also examined the Twist protein level in fresh primary ESCC and matched adjacent tissues from 10 patients using WB. Twist showed much higher expression levels in the tumor tissues than the matched adjacent tissues, and the expression was stronger in primary ESCC with LN metastasis than that without LN metastasis (Fig. 1B).
Nuclear expression of Twist correlated with cell migration and invasion ability. A highly invasive EC109 cell subline, EC109-P, was established for further study. WB and real time-PCR were used for the evaluation of Twist in ESCC cell lines. As shown in Figure 2A, a notably higher expression level of Twist was detected in EC109-P cells, a moderate level in EC109 cells, and the lowest level in EC9706 cells. The transwell assays demonstrated that EC109-P possessed an apparently higher invasion ability than the EC109 and EC9706 cells (Fig. 2B), and IF assays showed that the nuclear expression of Twist in EC109-P was markedly stronger than in the EC109 and EC9706 cells (Fig. 2C). These data indicated that the nuclear expression of Twist was correlated with the cell invasion.
The influence of the alteration Twist expression on the invasion of ESCC. Twist siRNA plasmid was transfected into cell line EC109-P, which has a high level of endogenous Twist expression. Both WB and real time PCR confirmed that Twist was significantly downregulated in the siRNA-transfected cells compared with the controls (Fig. 3A and B). The transwell assays showed that the knocking down of Twist notably reduced the invasion of the EC109-P cells ( Fig. 3C and D). To study whether Twist is involved in ESCC cell invasion further, a Twist overexpression plasmid was transfected into a cell line EC9706, which has a low endogenous Twist expression, and the expression of Twist was markedly upregulated (Fig. 4A and B). The transwell assays We established a highly invasive cell subline, EC109-P, using repetitive transwell assays. Consistent with the Twist expression in tissues, the most interesting results showed that the nuclear expression of Twist was markedly stronger in the highly invasive EC109-P cells compared with its parental EC109 cells, indicating that the nuclear expression of Twist was correlated with the migration and invasion ability of the cells. This phenomenon was further supported by the results that inhibiting Twist expression significantly suppressed the invasion of EC109-P cells and enhancing Twist expression showed the opposite effect in EC9706 cells.
VEGF-C plays significant roles in lymphatic vascular endothelium growth and the induction of selective hyperplasia of the lymphatic vasculature. 13,14 To clarify whether VEGF-C is involved in Twist-mediated LN metastasis in ESCC, WB and real time PCR were used to evaluate the co-expression of Twist and VEGF-C in cells transfected with siRNA and overexpression plasmids and corresponding controls. The results showed that knocking down of Twist can repress both the protein and mRNA expression of VEGF-C in EC109-P cells, whereas increased Twist expression had an opposite effect in EC9706 cells. Additionally, we also detected the VEGF-C expression in serial sections from 168 ESCC patients, and the results showed that the presence of high Twist expression in the nucleus correlated significantly with strong VEGF-C expression. Moreover, the regression analysis showed that patients with higher Twist in the nucleus and VEGF-C co-expression had a greater risk of developing lymph the regression analysis results suggested that patients with higher nuclear Twist levels and VEGF-C expression had a greater risk of developing lymph node metastasis (OR = 2.53, p < 0.01 vs. OR = 4.37, p < 0.001, respectively; Binary Logistic analysis).

Discussion
ESCC is one of the most malignant cancers and had a large prevalence worldwide. 1,2 As lymph node metastasis is a critical factor in determining ESCC prognosis, to understand the mechanisms of lymphatic metastasis, it is necessary for providing better diagnostic and therapeutic strategies for ESCC. Although several lines of evidence have confirmed the correlation of Twist with cancer progression, especially in LN metastasis, the mechanisms required further study.
In this study, we demonstrated for the first time that a nuclear location of Twist played an important role in ESCC lymphatic metastasis. The result of IHC of ESCC tissues from 168 patients showed that 60.7% of the samples possessed a nuclear localization of Twist, whereas 35.1% was in the cytoplasm. The cytoplasmic expression of Twist did not correlate with any of the clinicopathological data. In contrast, Twist expression in the nucleus was statistically correlated with LN metastasis, indicating that Twist expression in the nucleus was critical for the lymph node metastasis of ESCC. WB also showed that Twist presented notably elevated expression, especially in primary tumor tissues, with LN metastasis compared with matched adjacent tissues. The associations of Twist expression with clinicopathological parameters were evaluated by Kruskal-Wallis H test. *p < 0.05 was considered statistically significant.
168 ESCC patients, as described in our previous study in reference 25. Briefly, the endogenous peroxidase activity was blocked using 3% H 2 O 2 for 10 min at room temperature. The sections were incubated with Twist (1:500, Abcam) and VEGF-C (1:250, Abcam) antibodies in PBS overnight at 4°C. Sections incubated in PBS with anti-mouse/goat IgG (1:2,000, Zhongshan) but without primary antibody were used as negative controls. After three washes with PBS-T, the sections were incubated with anti-mouse/ node metastasis. Because Twist has been demonstrated to be a key transcription factor which induces the epithelial-mesenchymal transition, [5][6][7] we speculated that Twist accumulation in the nucleus and the alteration of VEGF-C might be related to its transcriptional activity.
In conclusion, Twist nuclear accumulation can promote LN metastasis and regulate this metastasis partly through altering VEGF-C expression in both ESCC tissues and cell lines. The combination of Twist and VEGF-C detection could be help in predicting the risk of LN metastasis for ESCC patients. Further experiments, such as luciferase reporter assays and chromatin immunoprecipitation, should be utilized to obtain more information about the precise mechanism of Twist and VEGF-C which is involved in the progression of ESCC LN metastasis.

Tissues.
A total of 168 primary Paraffinembedded tissues were obtained from ESCC patients, ranging from 37-to 77-yold (58.9 ± 9.8), who underwent surgery in Xijing Hospital of Digestive Diseases, Xian, China, from November 2008 to May 2011. Fresh surgical tissues from 10 ESCC patients (7 males and 3 females) who had undergone esophagectomy in our hospital between February and September of 2010 were stored at -80°C. All of the patients underwent transthoracic esophagectomy with three-field or two-field lymphadenectomy, and were confirmed by clinical and pathological diagnosis. Full clinical history records of these patients, including age, gender, location, T stage and LN metastasis, were also collected. None of the patients received preoperative chemotherapy or radiotherapy. Agreements for the tissue use were signed with informed consent by all of the patients and this study was approved by the Ethical Committee of Xijing Hospital. IHC staining. The IHC staining was performed to test the Twist and VEGF-C expression in serial sections of tissues from  was incubated in 5% fat-free milk for 1 h. The primary Twist antibody (1:500, Abcam), VEGF-C (1:500, Abcam) and β-actin (1:2,000, Sigma) were added. After three washes with PBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-goat or mouse secondary Ab (1:3,000; Sigma-Aldrich). Protein bands were detected using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). Each blot was repeated three times. Total RNA extraction and real-time PCR. Total tissue RNA was isolated from the cell lines using the TRIzol reagent (Invitrogen) as recommended by the manufacturer, and DNase was used to block the contamination of genomic DNA. Reverse transcription reactions were performed with 2 mg RNA using the Prime Script RT reagent kit and SYBR premix Ex Taq (Takara) with the following program: initial denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 15 sec, 56°C for 20 sec and 72°C for 15 sec. GAPDH was used as the housekeeping gene. The forward (F) and reverse (R) primer sequences were as follows: Twist: F, 5'-GGC CAG GTA CAT CGA CTT CC-3' and R, 5'-CCG CTC GTG AGC CAC ATA-3'; VEGF-C: F, 5'-TCA GGC AGC GAA CAA GAC-3' and R, 5'-GCA TCC GAG GAA AAC A-3'; GAPDH: F, 5'-GCA CCG TCA AGG CTG AGA AC-3' and R, 5'-TGG TGA AGA CGC CAG TGG A-3'. Each reaction was performed in triplicate.
ESCC cell lines and construction of a highly invasive EC109 subline. The human ESCC cell lines EC109 and EC9706 were obtained from the Chinese Academy of Medical Science. 26 The cells were cultured in RPMI-1640 medium (GIBCO) containing 10% heat-inactivated fetal calf serum (GIBCO) in a humidified incubator at 37°C in the presence of 5% CO 2 . The highly invasive EC109 cell subline was constructed as our previous report in reference 27, and described mainly below: EC109 cells in 1 ml serum-free 1640 were placed in the top chamber of an 8-μm-pore transwell (Corning) coated with 200 mg/ml Matrigel (BD Biosciences), the underlayer well was filled with 2.0 mL 1640 medium with 20% serum. After cultured for 24 h, the cells on the lower membrane were collected and expanded for fourround isolations.
Protein collection and western blot analysis. The protein in fresh tissues and cell lines were extracted as described in our previous work in reference 25. After loading the proteins (30 μg) onto an 8% SDS-PAGE, electrophoresis was performed at 20 mA for 60 min under denaturing conditions, and the proteins were then transferred to a nitrocellulose membrane. The membrane coated with 200 mg/mL Matrigel and dried at 4°C overnight. Afterward, 5 × 10 4 cells were placed in the top chamber, and the cells migrating across the transwell membrane were stained with 1% crystal violet 24 h after placement. The migrated cell number was manually counted using a light microscope (Olympus BX51, Olympus) at 200x magnification for ten random fields in each well.
Statistical analysis. All of the significant differences were analyzed using the SPSS 16.0 software package. A Kruskal-Wallis H test was performed to analyze the IHC of Twist expression with clinical parameters. The one-way ANOVA was used for the analysis of three comparisons, including the results of the real time PCR and transwell assays. Correlations were assessed using bivariate correlation and regression analysis was performed using Binary Logistic analysis. A p value < 0.05 was considered statistically significant.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Acknowledgments
This work was supported by the grant from China Postdoctoral Science Foundation (20090461447) and National Natural Science Foundation of China (81172288). 30 min. The cells were incubated with the primary Twist antibody (1:500, Abcam) overnight at RT. Donkey anti-mouse IgG-Cy3 (Molecular Probes, Invitrogen) was used as the secondary antibody. Lastly, the cells were counterstained with DAPI (Sigma) and examined using a fluorescence microscope (Olympus BX51, Olympus).
Plasmids and transfection. Twist overexpression and siRNA plasmids used in our lab, have been demonstrated high efficiencies of up and downregulating Twist expression. 28 The Twist-pcDNA 3.1 plasmid was transfected into EC9706 cells and the Psilence-Twist plasmid was transfected into EC109-P cells. The control plasmids, pcDNA 3.1 and Psilence, were transfected into the corresponding cells. The cell lines were named Si-EC109-P, Con-EC109-P, Twi-EC9706 and Con-EC9706 respectively. WB and real time PCR were used to detect the Twist protein and mRNA expression, and transwell assays were performed to evaluate the invasion ability of these cell lines. Each was repeated in triplicate.
Transwell assays. Twenty-four-well polycarbonate transwell plates with 8 μm pores (Corning) were used to test the ESCC cell line migration and invasion. Briefly, for the transwell migration assays, tumor cells were placed in the top and bottom chambers at a concentration of 5 × 10 4 in DMEM medium with 5% FBS. For the invasion assays, the top chambers were