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Cell Biology International (2012) 36, 571–577 (Printed in Great Britain)
Relationship between TWIST expression and epithelial–mesenchymal transition of oesophageal squamous cell carcinoma
Yuan Gao, Xiao‑Yan Xuan, Hong‑Yan Zhang, Feng Wang, Qing‑Ru Zeng, Zhi‑Qiang Wang and Shan‑Shan Li1
Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Key Laboratory of Tumor Pathology of Henan Province, Zhengzhou 450052, Henan Province, Peoples Republic of China


We have investigated mRNA and protein expression of TWIST, Vimentin and E-cadherin in ESCC (oesophageal squamous cell carcinoma) and explored their relationship with tumour's infiltration and metastasis. RT–PCR (reverse transcriptase–PCR) was used to evaluate mRNA expression of TWIST, E-cadherin and Vimentin in 40 cases of ESCC. The protein expression of the genes was examined by immunohistochemical staining in each specimen. Expression of TWIST, E-cadherin and Vimentin mRNA and protein with clinicopathologic parameters were analysed. mRNAs of TWIST, Vimentin and E-cadherin were expressed in 75, 55 and 35% respectively of ESCC, i.e. significantly different from that in normal oesophageal mucosa (15, 0 and 85% respectively; P<0.01). In ESCC with LN (lymph node) metastasis, expression of TWIST and Vimentin mRNA, but not E-cadherin mRNA was significantly higher (100 and 83%) than in ESCC without LN metastasis (64 and 43%, P = 0.018) respectively. Levels of mRNA expression of the 3 genes followed similar patterns to their above-mentioned frequencies. Protein expression of TWIST, E-cadherin and Vimentin were observed in 70, 35 and 50% respectively of ESCC, which were significantly different from normal mucosa (15, 80 and 0%; P<0.001). In ESCC with LN metastasis, protein expression of TWIST and Vimentin, but not E-cadherin, were significantly higher (100 and 75%) than in ESCC without LN metastasis (61 and 39%). Protein expression of TWIST was positively correlated with Vimentin (r = 0.327, P = 0.039), but negatively correlated with E-cadherin (r = −0.633, P = 0.000). Thus, both mRNAs and proteins of TWIST and Vimentin were significantly overexpressed in ESCC, especially ESCC with LN metastasis. The mRNA and protein of E-cadherin were down-regulated in ESCC. These results suggest potential roles of TWIST as the promoter of tumour invasion and metastasis associated with down-regulation of E-cadherin.


Key words: E-cadherin, epithelial–mesenchymal transition (EMT), oesophageal squamous cell carcinoma (ESCC), TWIST, Vimentin

Abbreviations: EMT, epithelial–mesenchymal transition, ESCC, oesophageal squamous cell carcinoma, LN, lymph node, RT–PCR, reverse transcriptase–PCR

1To whom correspondence should be addressed (email lsspath@yahoo.com.cn).


1. Introduction

ESCC (oesophageal squamous cell carcinoma) is the sixth most common malignant tumour worldwide (Ribeiro et al., 1996). The incidence of ESCC is very high in China (Li and Lu, 1996). Effective treatment depends on early diagnosis, with which more than 90% of the patients can survive 5–10 years (Li and Lu, 1996). Deep invasion and metastasis remain the leading causes of death for ESCC patients. Therefore, prevention of metastasis is a key for ESCC therapy.

Metastasis is a complex process and various factors are involved in each step of metastasis (Eccles and Welch, 2007). Recent studies suggest that numerous genes and proteins essential for embryonic development are mutated or aberrantly expressed in a variety of human cancers, and associated with tumour metastasis (Gilles and Thompson, 1996; Hay, 1995; Monk and Holding, 2001). Most aggressive metastatic cancer cells exhibited a characteristic EMT (epithelial–mesenchymal transition) (Zajchowski et al., 2001; Thiery, 2003; Xue et al., 2003; Bates and Mercurio, 2005; Huber et al., 2005; Thiery and Sleeman, 2006). Cells that undergo EMT morphogenesis switch from an apical–basolateral, polarized epithelial phenotype to a spindle-shaped, fibroblast-like mesenchymal phenotype. A key feature in the initiation and execution of EMT is down-regulation of E-cadherin expression and up-regulation of Vimentin expression (Zajchowski et al., 2001; Thiery, 2003; Bates and Mercurio, 2005; Peinado et al., 2007).

Several EMT-induced regulators repress E-cadherin transcription via interaction with specific E-boxes of the proximal E-cadherin promoter (Yang et al., 2004). TWIST, a highly conserved bHLH (basic helix-loop-helix) transcription factor, has been recently identified as a developmental gene with a key role in E-cadherin repression and EMT induction (Yang et al., 2004). Furthermore, a loss of TWIST expression prevents the intravasation of metastatic tumour cells into the blood circulation (Yang et al., 2006). TWIST-enhanced cancer metastasis includes breast cancer, gastric cancer and HCC (hepatocellular carcinoma) (Yang et al., 2004, 2007; Lee et al., 2006).

Therefore, we conducted the present study to examine the clinical significance of TWIST mRNA and protein expression in ESCC and the correlation between TWIST and E-cadherin/Vimentin expression in ESCC.

2. Materials and methods

2.1. Patients and tumour samples

Fresh tissues were taken from tumour and normal oesophageal mucosa (resection margin) from 40 patients with ESCC between July 2007 and July 2008 at The First Affiliated Hospital of Zhengzhou University. The patients consisted of 25 men and 15 women, and aged from 40 to 75 years with mean age of 60.4 years. After extraction for mRNA, a portion of representative tumour and normal tissue were processed for routine haematoxylin and eosin stained slides. The diagnoses of all ESCC were confirmed histologically for all tumours. Tumour was classified and staged with World Health Organization criteria (Gabbert et al., 2000). Of 40 cases, 10, 19 and 11 were well, moderately and poorly differentiated ESCC respectively. About a quarter (11/40) of ESCC showed superficial invasion and rest (29/40) showed deep invasion. Out of 40 ESCC, 12 had metastasis in LN (lymph node). None of the patients received prior radiotherapy or chemotherapy. Informed consent was obtained from all of the patients, and the study was approved by the Research Ethics Committee of Zhengzhou University.

2.2. RT–PCR (reverse transcriptase–PCR)

Total RNA was isolated from cancer and normal oesophageal mucosa from all the cases with TRIzol® reagent (Invitrogen) and reverse-transcribed with a One Step RNA PCR kit (AMV) (Sangon Biotechnology). Briefly, 6 μl of total RNA was reverse-transcribed at 50°C and amplified for 35 cycles with an annealing temperature of 58°C for TWIST, 55°C for E-cadherin and 58°C for Vimentin for 1 min. Primer sequences for TWIST were 5′-GGGAGTCCGCAGTCTTACGA-3′ and 5′-AGACCGAGAAGGCGTAGCTG-3′. For E-cadherin, primers 5′-TCCAAAGCCTCAGGTCATAAACATC-3′ and 5′-CGCCTCCTTCTTCATCATAGTAATAAACG-3′ were used in the PCRs. For Vimentin, primers 5′-ATGTGGATGTTTCCAAGCCTGAC-3′ and 5′-GAGTGGGTATCAACCAGAGGGAGT-3′ were used in PCR. The housekeeping gene β-actin was used as an internal control. Primers 5′-GCAGCCGTGGCCATCTCTTGCTC-3′ and 5′-AACCGCGAGAAGATGACCCAGATC-3′ were used to generate β-actin PCR products. RT–PCR was performed in triplicate for all the experiments with the negative control in which no template (total RNA) was added. RT–PCR products underwent electrophoresis in 2% agarose gel containing ethidium bromide. The corresponding DNA bands were scanned and semi-quantitatively analysed with an image analysis system (Stratagene). The units for TWIST, E-cadherin and Vimentin cDNA as well as β-actin bands were used to represent the levels of their mRNA expression after normalization to β-actin expression.

2.3. Immunohistochemical staining and evaluation

Four-micron-thick sections were deparaffinized, heated in 10 mM citrate buffer solution for 15 min at 70°C for antigen retrieval, and then quenched with 3% H2O2 for 25 min. After blockage in 1% BSA for 25 min, the slides were incubated with primary antibody of TWIST (Santa Cruz Biotechnology; sc-15393, 1:100), E-cadherin (Santa Cruz Biotechnology; sc-7870, 1:200), or Vimentin (Santa Cruz Biotechnology; sc-6260, 1:100) at 4°C for overnight, followed by staining with a streptavidin-biotin peroxidase kit (Zhongshan Goldenbridge Biotechnology). The sections were visualized by incubating with diaminobenzidine tetrahydrochloride and counterstained with haematoxylin and eosin. Normal oesophageal epithelium, invasive lobular carcinoma of the breast and malignant melanoma were used as positive controls for E-cadherin, TWIST and Vimentin respectively. Negative controls were created by replacing the primary antibodies with PBS. Evaluation of immunohistochemistry was independently carried out by two observers, who were blinded to the conditions of the experiment. For TWIST and Vimentin, cytoplasmic immunoreactivity was scored by its extent and intensity. For E-cadherin, plasmalemmal immunoreactivity was scored by its extent and intensity. Staining intensity was graded as follows (Wang et al., 2000): negative (0), weak (1), moderate (2) and strong (3). Staining extent was rated according to the percentage of positive cells. Samples with no stained tumour cells were rated as 0, those with <10% of stained tumour cells were rated as 1, those 11–50% of stained tumour cells were rated as 2, those with 50–75% of stained tumour cells were rated as 3 and those with >75% of stained tumour cells were rated as 4 (Wang et al., 2000). The score of staining intensity multiplied by the score of extent equals an overall staining score. An overall staining score of 0–3 and >3 were regarded as negative (−) and positive (+) protein expression respectively.

2.4. Statistical analysis

All data were analysed with SPSS version 13.0 statistical package (Shengce Software Ltd). The differences in positive rates and means were analysed by χ2 and t test respectively. The relationship of 2 variables was analysed by correlation analysis. P<0.05 was defined as statistically significant.

3. Results

3.1. Expressions of TWIST, E-cadherin and Vimentin mRNA in ESCC

Electrophoresis of RT–PCR products demonstrated amplified cDNA fragments at expected sizes, 277 bp for TWIST, 464 bp for E-cadherin, 544 bp for Vimentin and 384 bp for β-actin (Figure 1). The frequency of TWIST mRNA expression was observed in 75% (30/40) of ESCC, significantly higher than that in normal oesophageal mucosa [15% (6/40), P<0.01, Table 1]. Vimentin mRNA expression showed a similar pattern, 55% (22/40) in ESCC but none (0/40) in normal oesophageal mucosa (P<0.01). However, E-cadherin mRNA was expressed in a reverse pattern, 35% (14/40) in ESCC and 85% (34/40) in normal oesophageal mucosa (P<0.01, Table 1). In 12 cases of ESCC with LN metastasis, the frequency of TWIST mRNA expression was 100% (12/12), significantly higher than that in ESCC without LN metastasis [64.3% (18/28), P = 0.017, Table 1]. So was Vimentin mRNA expression, 83.3% (10/12) in ESCC with LN metastasis and 42.9% (12/28) in ESCC without LN metastasis (P = 0.018, Table 1). Vimentin mRNA was much more frequently expressed in the group of superficial layer than the group of deep layer (P = 0.030, Table 1). Expression of E-cadherin mRNA did not show correlation with tumour invasion, tumour differentiation and LN metastasis (Table 1).


Table 1 TWIST, E-cadherin, vimentin mRNA expression in relation to clinicopathological features in ESCC

TWIST mRNA E-cadherin mRNA Vimentin mRNA
Measurements n + (%) P TWIST/β-actin (±S) P + (%) P E-cadherin/β-actin (±S) P + (%) P Vimentin/β-actin (±S) P
Clinicopathologic features
    Normal 40 34 6 (15.0) 0.000 0.417±0.157 0.003 6 34 (85.0) 0.000 1.372±0.200 0.000 40 0 (0.00) 0.000 0.000
    ESCC 40 10 30 (75.0) 0.710±0.216 26 14 (35.0) 1.140±0.155 18 22 (55.0) 1.186±0.126
Tumour differentiation
    Well 10 4 6 (60.0) 0.259 0.532±0.183 0.072 4 6 (60.0) 0.122 1.192±0.156 0.402 7 3 (30.0) 0.056 1.083±0.215 0.089
    Moderate 19 5 14 (73.7) 0.746±0.186 13 6 (31.6) 1.130±0.147 9 10 (52.6) 1.160±0.118
    Poor 11 1 10 (90.9) 0.765±0.234 9 2 (18.2) 1.015±0.191 2 9 (81.8) 1.250±0.072
Tumour invasion
    Superficial layer 11 5 6 (54.5) 0.066 0.617±0.137 0.245 5 6 (54.5) 0.110 1.072±0.174 0.236 8 3 (27.3) 0.030 1.213±0.111 0.010
    Deep layer 29 5 24 (82.8) 0.733±0.228 21 8 (27.6) 1.178±0.140 10 19 (65.5) 1.020±0.085
LN metastases
    Yes 12 0 12 (100.0) 0.017 0.801±0.195 0.006 8 4 (33.3) 0.885 1.175±0.119 0.614 2 10 (83.3) 0.018 1.242±0.077 0.009
    No 28 10 18 (64.3) 0.591±0.187 18 10 (35.7) 1.126±0.171 16 12 (42.9) 1.106±0.143



Semi-quantitative analyses showed the level of mRNA expression of these three genes exhibited the similar patterns as their frequencies mentioned above: for TWIST, 0.710±0.216 in ESCC and 0.417±0.157 in normal oesophageal mucosa (P = 0.003); for E-cadherin, 1.140±0.155 in ESCC and 1.372±0.200 in normal oesophageal mucosa (P<0.001); for Vimentin, 1.186±0.126 in ESCC and zero in normal oesophageal mucosa (P<0.001, Table 1). In 12 cases of ESCC with LN metastasis, the average of TWIST mRNA level was much higher (0.801±0.195) than that in ESCC without LN metastasis (P = 0.006, Table1). So was the Vimentin mRNA level, 1.242±0.077 in ESCC with LN metastasis and 1.106±0.143 in ESCC without LN metastasis (P = 0.009, Table 1). The mRNA level of E-cadherin was similar in both ESCC with LN metastasis (1.175±0.119) and ESCC without LN metastasis (1.126±0.171, P = 0.641, Table 1). Among the clinicopathologic parameters, Vimentin mRNA level was associated with ESCC with deep invasion (P = 0.01). The levels of TWIST and E-cadherin mRNAs were not correlated with tumour differentiation, tumour invasion, or LN metastasis (E-cadherin only, P>0.05, Table 1).

3.2. Correlation of mRNA expression of TWIST with that of E-cadherin/Vimentin in ESCC

To explore the correlation of TWIST and E-cadherin or Vimentin mRNA expression in ESCC, the frequencies of their mRNA expressions were compared. Analysis showed that mRNA expression of TWIST was positively correlated with that of Vimentin (r = 0.406, P = 0.009), but negatively correlated with that of E-cadherin in ESCC (r = −0.666, P<0.001, Table 2).


Table 2 Correlation of mRNA expression of TWIST with that of E-cadherin/vimentin in ESCC

E-cadherin Vimentin
TWIST + Total r P + Total r P
+ 5 25 30 −0.666 0.000 20 10 30 0.406 0.009
9 1 10 2 8 10
Total 14 26 40 22 18 40



3.3. Expressions of TWIST, E-cadherin and Vimentin protein in ESCC

Since expression of mRNA does not always match with translation of protein, the protein expression of TWIST, E-cadherin and Vimentin was explored in ESCC. The results showed that TWIST protein was expressed in 70% (28/40) of ESCC, significantly higher than that in normal oesophageal mucosa [15% (6/40), P<0.001, Figure 2, Table 3]. Vimentin protein followed a similar pattern, 50% (20/40) in ESCC and zero (0/40) in normal oesophageal mucosa (P<0.001, Figure 2, Table 3). E-cadheirn protein was expressed in a reverse pattern when compared with that of TWIST, 35% (14/40) in ESCC but 80% (32/40) in normal oesophageal mucosa (P<0.001, Figure 2, Table 3). In 12 ESCC with LN metastasis, expression of TWIST protein was expressed in 100% (12/12) of them, significantly higher than that in ESCC without LN metastasis [60.7% (16/40), P = 0.007, Table 3]. So was Vimentin protein, 75% (9/12) in ESCC with LN metastasis and 39.3% (11/28) in ESCC without LN metastasis (P = 0.038, Table 3). No significant association of E-cadherin protein expression with tumour's grade, differentiation, and depth of tumour invasion (P>0.05, Table 3).


Table 3 TWIST, E-cadherin and vimentin protein expressions in relation to clinical pathological characteristics in ESCC

TWIST E-cadherin Vimentin
Clinicopathologic features n + (%) χ2 P +(%) χ2 P +(%) χ2 P
Normal 40 34 6 (15.0) 24.757 0.000 8 32 (80.0) 16.573 0.000 40 0 (0) 22.661 0.000
ESCC 40 12 28 (70.0) 26 14 (35.0) 20 20 (50.0)
Tumour differentiation
Well 10 4 6 (60.0) 0.505 0.777 4 6 (60.0) 4.213 0.122 7 3 (30.0) 3.925 0.140
Moderate 19 6 13 (68.4) 13 6 (31.6) 10 9 (47.4)
Poor 11 2 9 (81.8) 9 2 (18.2) 3 8 (72.7)
Tumour invasion
Superficial layer 11 5 6 (54.5) 1.726 0.189 5 6 (54.5) 2.548 0.110 8 3 (27.3) 3.135 0.077
Deep layer 29 7 22 (75.9) 21 8 (27.6) 12 17 (58.6)
LN metastases
Yes 12 0 12 (100.0) 7.347 0.007 8 4 (33.3) 0.021 0.885 3 9 (75.0) 4.286 0.038
No 28 12 16 (60.7) 18 10 (35.7) 17 11 (39.3)



3.4. Correlation of protein expression of TWIST with that of E-cadherin/Vimentin in ESCC

Of 28 cases of ESCC expressed TWIST protein, only 4 cases expressed E-cadherin protein and 17 cases expressed Vimentin protein. The protein expression of TWIST was positively correlated with that of Vimentin (r = 0.327, P = 0.039, Table 4), but negatively correlated with that of E-cadherin protein (r = −0.633, P<0.001, Table 4).


Table 4 Correlation of protein expression of TWIST with that of E-cadherin/vimentin in ESCC

E-cadherin Vimentin
TWIST + Total r P + Total r P
+ 4 24 28 -0.663 0.000 17 11 28 0.327 0.039
10 2 12 3 9 12
Total 14 26 40 20 20 40



4. Discussion

A tumour's invasion is usually the prerequisite for metastasis, which remains the biggest hurdle to have cancer patient cured (Eccles and Welch, 2007). There still are many unknowns to be solved in the complex process of tumour metastasis. However, more and more evidence suggest a role of EMT in this process since it would weaken E-cadherin-dependent intercellular adhesion and enhance motility of tumour cells (Huber et al., 2005; Thiery and Sleeman, 2006). During EMT, many molecules undergo significant changes, including down-regulation of E-cadherin responsible for the loss of cell–cell adhesion; up-regulation of matrix-degrading proteases and mesenchymal-related proteins such as Vimentin and N-cadherin; and up-regulation and/or nuclear translocation of transcription factors underlying the specific gene program of EMT, such as Snail, Slug, ZEB and TWIST (Thiery and Sleeman, 2006).

Ectopic expression of TWIST results in loss of E-cadherin-mediated cell–cell adhesion, activation of mesenchymal markers and induction of cell motility. These findings suggest that TWIST contributes to tumour invasiveness and metastasis by promoting an EMT (Yang et al., 2004). Recently, up-regulation of TWIST has been reported in several human cancers (Yang et al., 2004; Hoek et al., 2004; Elias et al., 2005; Kwok et al., 2005; Entz-Werle et al., 2005; Kyo et al., 2006; Sasaki et al., 2009). Simultaneous overexpression of TWIST (36–60%) and down-regulation of E-cadherin (44–74%) were also reported in a variety of human cancers (Nakanishi et al., 1997; Krishnadath et al., 1997; Richmond et al., 1997; Kyo et al., 2006; Zhang et al., 2007; Yuen et al., 2007a; Sasaki et al., 2009; Pecina-Slaus et al., 2012).

In this study, both mRNA and protein of TWIST were significantly overexpressed in ESCC tissues as compared with their paired normal oesophageal mucosa. The mRNA and protein of Vimentin were expressed in 55% (22/40) and 50% (20/40) of ESCC respectively, but in none of their paired normal oesophageal mucosa. These results suggest TWIST and Vimentin may serve as the potential tumour markers for the diagnosis of ESCC.

TWIST gene is also known as a potential oncogene (Mastro et al., 1999). It can inhibit myc- and p53-dependent apoptosis in mouse embryonic fibroblasts as well as NF-κB (nuclear factor κB) pathway-dependent apoptosis (Mastro et al., 1999; Sosic and Olson, 2003). Our data showed that mRNA and protein of TWIST were expressed in 15% (6/40) of normal oesophageal mucosa. This result may represent an early neoplastic transformation of normal oesophageal epithelium caused by TWIST.

The most interesting finding on TWIST is that its overexpression is closely associated with tumour metastasis in human cancers (Yuen et al., 2007b). Up-regulation of TWIST has been associated with high incidence of distant metastasis whereas down-regulation of E-cadherin expression has been associated with high incidence of LN metastasis in ESCC (Nakanishi et al., 1997; Setoyama et al., 2007; Yuen et al., 2007a). In this study, not only the frequencies and levels of mRNAs but also the proteins of TWIST and Vimentin mRNA were significantly higher in ESCC with LN metastasis than in those without LN metastasis. These results are consistent with those reported for other human cancers, suggesting their potential roles as the biomarker to predict the metastasis of ESCC. It also indicates that TWIST and Vimentin may be involved in promoting the metastasis of ESCC.

The mechanism with which TWIST promote tumour invasion and metastasis is not completely understood. It was reported that Mesenchyme Forkhead 1 [FOXC2 (forkhead box C2)] which induced by TWIST, Snail, Goosecoid and TGF-β1 (transforming growth factor-β1) plays a central role in promoting invasion and metastasis in human basal-like breast cancers (Mani et al., 2007). In addition, TWIST plays a key role in E-cadherin repression and EMT induction (Yang et al., 2004). In epithelial cells, high expression of TWIST is associated with a loss in epithelial markers, including E-cadherin and β-catenin (Yang et al., 2004). Instead, these cells express mesenchymal markers, including Vimentin and N-cadherin. In fact, inverse correlation between high TWIST and reduced E-cadherin expression has been found in liver, bladder and prostate cancer cells (Lee et al., 2006; Kyo et al., 2006; Yuen et al., 2007a). Our data also showed the mRNA and protein expression of TWIST was negatively correlated with that of E-cadherin but positively correlated with that of Vimentin. These results are consistent with those in the reports and suggest that TWIST may promote tumour metastasis through down-regulation of E-cadherin and/or up-regulation of Vimentin in ESCC.

Author contribution

Shan-Shan Li conceived and designed the experiments. Yuan Gao, Xiao-Yan Xuan, Qing-Ru Zeng, Zhi-Qiang Wang performed the experiments. Shan-Shan Li and Xiao-Yan Xuan contributed reagents/materials/analysis tools. Yuan Gao, Hong-Yan Zhang and Feng Wang analysed the data. Yuan Gao wrote the paper.

Acknowledgments

The work was completed in the Key Laboratory of Tumor Pathology of He'nan Province, P.R. China. We thank Professor Zhang Li-rong (College of Basic Medical, Zhengzhou University, Zhengzhou, Henan, China) for giving us advice.

Funding

This work was supported by the National Natural Science Foundation of China [grant number 81071970] and the Medical Science and Technology Program of He'nan Province [grant number 200801006].

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Received 22 March 2010/18 January 2012; accepted 23 February 2012

Published as Cell Biology International Immediate Publication 23 February 2012, doi:10.1042/CBI20100195


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)