Twist-1基因表达在食管鳞状细胞癌中的临床病理学意义
摘要
目的:应用RNAi,Western blot,Real-time PCR,MS-PCR和免疫组化等方法研究Twist-1基因蛋白表达在食管鳞状细胞癌(Esophageal Squamous Cell Carcinoma,ESCC)中的临床病理学意义。
材料和方法:食管鳞状细胞癌(ESCC)167例及正常食管组织34例共201例病理组织标本选自韩国三星医疗中心病理科1995-2008年的存档蜡块标本。所有标本均制作成组织芯片(TMA,tissue microarray)。另外,我们选择近几年已发表论文中最常使用的几种Twist抗体,应用RNAi技术、Western blot和免疫细胞化学方法在10种胃癌细胞株中进行Twist-1蛋白表达的对比检测,从而筛选出特异性最好的抗体应用于上述食管病变蜡块标本的免疫组化染色,同时进行Real-time PCR和Twist-1基因启动子区甲基化(Methylation Specific PCR,MS-PCR)检测,探讨Twist-1基因和蛋白在ESCC患者中的表达情况,并分析其临床病理学意义及其机制。
结果:与其它Twist抗体比较,单克隆抗体Twist2C1a(ab50887)具有背景染色少、抗原特异性高的特点,因此是比较可靠的Twist-1抗体。单克隆抗体Twist2C1a(ab50887)蛋白免疫组化染色结果表明,Twist-1蛋白阳性表达部位主要是食管鳞癌细胞核,Twist-1蛋白在食管鳞癌中的阳性表达率较高(86/167,51.5%),而在正常食管鳞状上皮组织中则较低(9/34,26.5%),且差异具有显著性(p=0.008＜0.01)。对ESCC患者的临床病理学数据的分析结果表明,Twist-1蛋白过表达预示食管鳞癌患者的不良预后,即5年生存率明显降低,在Cox-比例风险模型统计单变量(RR=3.019,p=0.000)和多变量(RR=3.131,p=0.000)分析中均有意义。但与患者年龄、性别、肿瘤大小、分期、有无淋巴结转移、癌细胞分化情况以及放化疗等不相关(p＞0.05)。另外,根据免疫组化染色结果选取Twist-1蛋白高表达和低表达食管鳞癌蜡块组织各7例以及正常食管鳞状上皮蜡块组织5例,应用Real-time PCR检测标本的mRNA表达水平,结果表明各病例中的mRNA表达水平与免疫组化结果相一致,即免疫组化Twsit-1蛋白过表达病例,其mRNA表达水平也明显增高。同样,MS-PCR结果也证明,Twist-1蛋白低表达病例表现为Twist-1基因启动子区的高度甲基化,而Twist-1蛋白强阳性病例表现为Twist-1基因启动子区无甲基化或甲基化程度明显较弱。
结论:单克隆抗体Twist2C1a(ab50887)的抗原特异性要优于其它Twist抗体,可用于各种病变组织中Twist-1蛋白的检测,而且Twist-1蛋白检测可以作为食管鳞癌的预后判定指标之一。Twist-1蛋白在食管鳞癌中的过表达机制与Twist-1基因启动子区甲基化密切相关,并有望成为食管鳞癌治疗的新靶点。
Objectives: To investigate the clinicopathological significance of Twist-1 gene expression in esophageal squamous cell carcinoma (ESCC) by RNAi, Western blot, Real-time PCR, MS-PCR, immunohistochemical staining and other techniques.
Materials and Methods: Total 201 cases of specimen, including 167 of ESCC and 34 of normal esophagus, were selected from Department of Pathology, Samsung Medical Center (South Korea) in the period of 1995-2008, and the tissue microarray (TMA) was made by using all above tissues. To select the best antibodies of Twist-1 protein, four kinds of different Twist antibodies were tested in 10 gastric cell lines by RNAi, Western blot analysis and cell block immunocytochemistry (ICC), and the best one was used to investigate the protein expression of Twist-1 in ESCC and normal esophagus by immunohistochemical staining. The correlation between Twist-1 over-expression and clinicolpathological parameters of ESCC was also analyzed. Then the Twist-1 mRNA level and DNA methylation status of Twist-1 gene promoter were detected by Real-time PCR and Methylation Specific PCR (MS-PCR) for exploring the mechanism of Twist-1 protein over-expression in ESCC.
Results: Monoclonal antibody, Twist2Cla (ab50887), was the best antibody for detection of Twist-1 antigen specificity with low level of background. Twist-1 protein expression level was significantly increased in ESCC than in normal esophagus (86/167, 51.5% versus 9/34, 26.5%, p =0.008<0.01). The over-expression of Twist-1 in ESCC indicated the poor-prognosis (low 5 years survival) (p<0.001), however, there was no significant difference between Twist-1 protein expression and the clinicopathological parameters, including age, sex, lymph node metastasis, tumor size,stage, differentiation, chemical therapy and radiation therapy (p>0.05). Also the correlation of Twist-1 over-expression with overall survival rate of the ESCC patients was analyzed, and it showed the statistical significance in univariate analysis (RR=3.019, p=0.000) and multivariate analysis (RR=3.131, p=0.000) by the Cox-proportional hazards regression analysis (Figure 3). According to the IHC result, 7 ESCC cases with Twist-1 over-expression, 7 ESCC cases with Twist-1 lower expression, and 5 cases of normal esophagus were selected; the mRNA expression level of the blocks was detected by RT-PCR. It showed that Twist-1 mRNA expression level is also significant high in the cases with Twist-1 strong positivity by IHC. The result of MS-PCR showed that the hypermethylation of Twist-1 might be corrrelated with the low expression of Twist-1 protein in esophageal lesions, whereas the unmethylation of Twist-1 might be the cause of Twist-1 high expression.
Conclusions: The monoclonal antibody Twist2Cla (ab50887) was the best one for detection of Twist-1 protein. Twist-1 was highly expressed in ESCC, and the positive expression was strongly associated with patients' survival with a statistical significance. Meanwhile, the mechanism of Twist-1 high expression in ESCC was correlated with the methylation status of Twist-1 gene promoter, and Twist-1 was involved in the progression of ESCC and could be the molecular target for ESCC therapy.
材料和方法:食管鳞状细胞癌(ESCC)167例及正常食管组织34例共201例病理组织标本选自韩国三星医疗中心病理科1995-2008年的存档蜡块标本。所有标本均制作成组织芯片(TMA,tissue microarray)。另外,我们选择近几年已发表论文中最常使用的几种Twist抗体,应用RNAi技术、Western blot和免疫细胞化学方法在10种胃癌细胞株中进行Twist-1蛋白表达的对比检测,从而筛选出特异性最好的抗体应用于上述食管病变蜡块标本的免疫组化染色,同时进行Real-time PCR和Twist-1基因启动子区甲基化(Methylation Specific PCR,MS-PCR)检测,探讨Twist-1基因和蛋白在ESCC患者中的表达情况,并分析其临床病理学意义及其机制。
结果:与其它Twist抗体比较,单克隆抗体Twist2C1a(ab50887)具有背景染色少、抗原特异性高的特点,因此是比较可靠的Twist-1抗体。单克隆抗体Twist2C1a(ab50887)蛋白免疫组化染色结果表明,Twist-1蛋白阳性表达部位主要是食管鳞癌细胞核,Twist-1蛋白在食管鳞癌中的阳性表达率较高(86/167,51.5%),而在正常食管鳞状上皮组织中则较低(9/34,26.5%),且差异具有显著性(p=0.008＜0.01)。对ESCC患者的临床病理学数据的分析结果表明,Twist-1蛋白过表达预示食管鳞癌患者的不良预后,即5年生存率明显降低,在Cox-比例风险模型统计单变量(RR=3.019,p=0.000)和多变量(RR=3.131,p=0.000)分析中均有意义。但与患者年龄、性别、肿瘤大小、分期、有无淋巴结转移、癌细胞分化情况以及放化疗等不相关(p＞0.05)。另外,根据免疫组化染色结果选取Twist-1蛋白高表达和低表达食管鳞癌蜡块组织各7例以及正常食管鳞状上皮蜡块组织5例,应用Real-time PCR检测标本的mRNA表达水平,结果表明各病例中的mRNA表达水平与免疫组化结果相一致,即免疫组化Twsit-1蛋白过表达病例,其mRNA表达水平也明显增高。同样,MS-PCR结果也证明,Twist-1蛋白低表达病例表现为Twist-1基因启动子区的高度甲基化,而Twist-1蛋白强阳性病例表现为Twist-1基因启动子区无甲基化或甲基化程度明显较弱。
结论:单克隆抗体Twist2C1a(ab50887)的抗原特异性要优于其它Twist抗体,可用于各种病变组织中Twist-1蛋白的检测,而且Twist-1蛋白检测可以作为食管鳞癌的预后判定指标之一。Twist-1蛋白在食管鳞癌中的过表达机制与Twist-1基因启动子区甲基化密切相关,并有望成为食管鳞癌治疗的新靶点。
Objectives: To investigate the clinicopathological significance of Twist-1 gene expression in esophageal squamous cell carcinoma (ESCC) by RNAi, Western blot, Real-time PCR, MS-PCR, immunohistochemical staining and other techniques.
Materials and Methods: Total 201 cases of specimen, including 167 of ESCC and 34 of normal esophagus, were selected from Department of Pathology, Samsung Medical Center (South Korea) in the period of 1995-2008, and the tissue microarray (TMA) was made by using all above tissues. To select the best antibodies of Twist-1 protein, four kinds of different Twist antibodies were tested in 10 gastric cell lines by RNAi, Western blot analysis and cell block immunocytochemistry (ICC), and the best one was used to investigate the protein expression of Twist-1 in ESCC and normal esophagus by immunohistochemical staining. The correlation between Twist-1 over-expression and clinicolpathological parameters of ESCC was also analyzed. Then the Twist-1 mRNA level and DNA methylation status of Twist-1 gene promoter were detected by Real-time PCR and Methylation Specific PCR (MS-PCR) for exploring the mechanism of Twist-1 protein over-expression in ESCC.
Results: Monoclonal antibody, Twist2Cla (ab50887), was the best antibody for detection of Twist-1 antigen specificity with low level of background. Twist-1 protein expression level was significantly increased in ESCC than in normal esophagus (86/167, 51.5% versus 9/34, 26.5%, p =0.008<0.01). The over-expression of Twist-1 in ESCC indicated the poor-prognosis (low 5 years survival) (p<0.001), however, there was no significant difference between Twist-1 protein expression and the clinicopathological parameters, including age, sex, lymph node metastasis, tumor size,stage, differentiation, chemical therapy and radiation therapy (p>0.05). Also the correlation of Twist-1 over-expression with overall survival rate of the ESCC patients was analyzed, and it showed the statistical significance in univariate analysis (RR=3.019, p=0.000) and multivariate analysis (RR=3.131, p=0.000) by the Cox-proportional hazards regression analysis (Figure 3). According to the IHC result, 7 ESCC cases with Twist-1 over-expression, 7 ESCC cases with Twist-1 lower expression, and 5 cases of normal esophagus were selected; the mRNA expression level of the blocks was detected by RT-PCR. It showed that Twist-1 mRNA expression level is also significant high in the cases with Twist-1 strong positivity by IHC. The result of MS-PCR showed that the hypermethylation of Twist-1 might be corrrelated with the low expression of Twist-1 protein in esophageal lesions, whereas the unmethylation of Twist-1 might be the cause of Twist-1 high expression.
Conclusions: The monoclonal antibody Twist2Cla (ab50887) was the best one for detection of Twist-1 protein. Twist-1 was highly expressed in ESCC, and the positive expression was strongly associated with patients' survival with a statistical significance. Meanwhile, the mechanism of Twist-1 high expression in ESCC was correlated with the methylation status of Twist-1 gene promoter, and Twist-1 was involved in the progression of ESCC and could be the molecular target for ESCC therapy.
引文
[1] Yuen H F, Chan Y P, Chan K K, et al. Id-1 and Id-2 are markers for metastasis and prognosis in oesophageal squamous cell carcinoma. Br J Cancer, 2007, 97(10): 1409-1415
[2] Ries LA G, Eisner M P, Kosary C L, et al. SEER Cancer Statistics Review, 1975-2000, National Cancer Institute.
[3] Lieberman M D, Franseschi D, Marsan B, et al. Esophageal carcinoma: the unusual variants. J Thorac Cardiovasc Surg, 1994,108:1138-1146
[4] Lam A K. Molecular biology of esophageal squamous cell carcinoma. Crit Rev Oncol Hematol, 2000,33(2):71-90
[5] Blot W J. Esophageal cancer trends and risk factors. Semin Oncol, 1994,21: 403-410
[6] Marger R S, Marger D. Carcinoma of the esophagus and tylosis: a lethal genetic combination. Cancer, 1993,72:17-19
[7] Nakakubo Y, Hida Y, Miyamoto M, et al. The prognostic significance of RCAS1 expression in squamous cell carcinoma of the oesophagus. Cancer Lett, 2002,177(1):101-105
[8] Ando N, Ozawa S, Kitagawa Y, et al. Improvement in the results of surgical treatment of advanced squamous esophageal carcinoma during 15 consecutive years. Ann Surg, 2000,232(2):225-232
[9] Adham M, Baulieux J, Mornex F, et al. Combined chemotherapy and radiotherapy followed by surgery in the treatment of patients with squamous cell carcinoma of the esophagus. Cancer, 2000, 89(10): 2144
[10] Sporn M B. The war on cancer. Lancet, 1996,347:1377-1381
[11] Karreth F, Tuveson D A. Twist induces an Epithelial-Mesenchymal transition to facilitate tumor metastasis, Cancer Biol Ther, 2004, 3(ll):1058-1059
[12] Christofori G. Changing neighbours, changing behaviour: cell adhesion molecule-mediated signalling during tumour progression. EMBO J, 2003, 22:2318-2323
[13] Chambers A F, Matrisian L M. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst, 1997, 89:1260-1270
[14] Janda E, Lehmann K, Killisch I, et al. Ras and TGFb cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol, 2002,156:299-313
[15] Thiery J P. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol, 2003,15:740-746
[16] Timmerman L A, Grego-Bessa J, Raya A, et al. Notchpromotes epithelial--mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev, 2004,18:99-115
[17] Lu Z, Ghosh S, Wang Z, et al. Down regulation of caveolin-1 function by EGF leadsto the loss of E-cadherin, increased transcriptional activity of b-catenin, and enhanced tumor cell invasion. Cancer Cell, 2003,4:499-515
[18] Bolos V, Peinado H, Perez-Moreno M A, et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci, 2003, 116: 499-511
[19] Yang J, Mani S A, Donaher J L, et al. Twist, a masterregulator of morphogenesis, plays an essential role intumor metastasis. Cell, 2004,117:927-939
[20] Lee M S, Lowe G N, Strong D D, et al. TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage. J Cell Biochem, 1999,75(4):566-577
[21] Li L, Cserjesi P, Olson E N. Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis. Dev Biol, 1995,172:280-292
[22] Sosic D, Richardson J A, Yu K, et al. Twist regulates cytokine gene expression through a negative feedback loop that represses NF-B activity. Cell, 2003,112: 169-180
[23] Hoek K, Rimm D L, Williams K R, et al. Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas. Cancer Res, 2004,64: 5270-5282
[24] Wang X, Ling M T, Guan X Y, et al. Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells. Oncogene, 2004,23: 474-482
[25] Kwok W K, Ling M T, Lee T W, et al. Up-regulation of TWIST in prostate cancer and its implication as a therapeutic target. Cancer Res, 2005,65: 5153-5162
[26] Dupont J, Fernandez A M, Glackin C A, et al. Insulinlike growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor. J Biol Chem, 2001,276:26699-26707
[27] Puisieux A, Valsesia-Wittmann S, Ansieau S, et al. A twist for survival and cancer progression. Br J Cancer, 2006,94(1): 13-17
[28] Kawana K Y, Kawana H, Yoshikawa Y, et al. Nasal immunization of mice with peptide having a cross-neutralization epitope on minor capsid protein L2 of human papillomavirus type 16 elicit (sic) systemic and mucosal antibodies. Vaccine, 2001,19:1496-1502
[29] Niu R F, Zhang L, Xi G M, et al. Up-regulation of Twist induces angiogenesis and correlates with metastasis in hepatocellular carcinoma. J Exp Clin Cancer Res,2007,26(3):385-394
[30] Yan Q Z, Xue Y G, Shuang H, et al. Expression and significance of TWIST basic helix-loop-helix protein over-expression in gastric cancer. Pathology, 2007 39(5):470-475
[31] Yuen H F, Chua C W, Chan Y P, et al. Significance of TWIST and E-cadherin expression in the metastatic progression of prostatic cancer. Histopathology, 2007,50(5):648-658
[32] Ohuchida K, Mizumoto K, Ohhashi S, et al. Twist, a novel oncogene, is upregulated in pancreatic cancer: clinical implication of Twist expression in pancreatic juice. Int J Cancer, 2007,120(8):1634-1640
[33] Sinicrope F A, Ruan S B, Cleary K R, et al. bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis. Cancer Res, 55:237-241
[34] Olek A, Oswald J, Walter J. A modified and improved method for bisulphate based cytosine methylation analysis. Nucleic Acids Res,1996,15(24):5064-5066
[35] Gripp K W, Zackai E H, Stolle C A. Mutations in the human TWIST gene. Hum Mutat, 2000;15(2):150-155
[36] Jan Y N, Jan L Y. Functional gene cassettes in development. Proc Natl Acad Sci USA, 1993,90: 8305-8307
[37] Thisse B, Stoetzel C, Messal M, et al. The twist gene:isolation of a Drosophila zygotic gene necessary for the establishment of dorso-ventral pattern. Nucleic Acids Res, 1987,15:3439-3453
[38] Park R E, Kosman D, Bier E, et al. The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev, 1992,6:1728-1739
[39] Leptin M. twist and snail as positive and negative regulators duringDrosophila mesoderm development. Genes Dev, 1991,5:1568-1576
[40] Maestro R, Dei Tos A P, Hamamori Y, et al. Twist is a potential oncogene that inhibits apoptosis. Genes Dev, 1999,13:2207-2217
[41] Rosivatz E, Becker I, Specht K, et al. Differential expression of the epithelial -mesenchymal transition regulators Snail, SIP1, and Twist in gastric cancer. Am J Pathol, 2002,161:1881-1891
[42] Martin T A, Goyal A, Watkins G, et al. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast cancer. Ann Surg Oncol, 2005,12(6):488-496
[43] Hoek K, Rimm D L, Williams K R, et al. Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas. Cancer Res, 2004 64(15):5270-5282
[44] Song L B, Liao W T, Mai H Q, et al. The clinical significance of twist expression in nasopharyngeal carcinoma. Cancer Lett, 2006,242(2):258-265
[45] Yuen H F, Chan Y P, Wong M L, et al. Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis. J Clin Pathol, 2007,60(5):510-514
[46] Furlong E E, Andersen E C, Null. B, et al. Patterns of gene expression during Drosophila mesoderm development. Science, 2001,293:1629-1633
[47] Chen Z F, Behringer R R. Twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev, 1995,9:686-699
[48] Soo K, O'Rourke M P, Khoo P L, et al. Twist function is required for the morphogenesis of the cephalic neural tube and the differentiation of the cranial neural crest cells in the mouse embryo. Dev Biol, 2002,247:251-270
[49] Meulemans D, Bronner-Fraser M. Gene-regulatory interactions in neural crest evolution and development. Dev Cell, 2004,7:291-299
[50] Maestro R, Dei Tos A P, Hamamori Y, et al. TWIST is a potential oncogene that inhibits apoptosis. Genes Dev, 1999,13:2207-2217
[51] Valsesia-Wittmann S, Magdeleine M, Dupasquier S, et al. Oncogenic cooperation between H-TWIST and the N-Myc overrides failsafe programs in cancer cells. Cancer Cell, 2004,6:625-630
[52] Sosic D, Richardson J A, Yu K, et al. TWIST regulates cytokine gene expression through a negative feedback loop that represses NF-kappa B activity. Cell, 2003,112:169-180
[53] Janda E, Lehmann K, Killisch I, et al. Ras and TGF[h] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol, 2002,156:299-313
[54] Batlle E, Sancho E, Franci C, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol, 2000, 2:84-89
[55] Cano A, Perez-Moreno M A, Rodrigo I, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.Nat Cell Biol, 2000,2:76-83
[56] Hajra K M, Chen D Y, Fearon E R. The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res, 2002,62:1613-1618
[57] Comijn J, Berx G, Vermassen P, et al. The twohanded E-box binding zinc finger protein SIP1downregulates E-cadherin and induces invasion. Mol Cell, 2001, 7:1267-1278
[58] Shiren S, Xiao X N, Yan Q Z, et al. Hypoxia-inducible factor-1a induces Twist expression in tubular epithelial cells subjected to hypoxia, leading to epithelial-to-mesenchymal transition. Kidney Int, 2009,March, 11:1-10
[1] Yang J, Mani S A, Donaher J L, et al. Twist, a masterregulator of morphogenesis, plays an essential role intumor metastasis. Cell, 2004,117:927-939
[2] Thisse B, Stoetzel C, El Messal M, et al. The twist gene: Isolation of a Drosophila zygotic gene necessary for the establishment of dorso-ventral pattern. Nucleic Acids Res, 1987,15:3439-3453
[3] Jan Y N, Jan L Y. Functional gene cassettes in development. Proc Natl Acad Sci USA, 1993,90: 8305- 8307
[4] Ip Y T, Park R E, Kosman D, et al. The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev,1992, 6: 1728-1739
[5] Leptin M. Twist and Snail as positive and negative r egulators during Drosophilamesoderm development. Genes Dev, 1991,5:1568-1576
[6] Li L, Cserjesi P, Olson E N. Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis. Dev Biol ,1995,172:280-292
[7] Bialek P, Kern B, Yang X, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell, 2004, 6: 423-435
[8] Sosic D, Richardson J A, Yu K, et al. Twist regulates cytokine gene expression through a negative feedback loop that represses NF-B activity. Cell, 2003,112: 169-180
[9] Pantke O A, Cohen Jr MM, Witkop Jr CJ, et al.The Saethre-Chotzen syndrome. Birth Defects OrigArti Ser,1975,11:190-225
[10] Chen Z F, Behringer R R. Twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev, 1995,9: 686-699
[11] Maestro R, Dei Tos AP, Hamamori Y, et al. Twist is a potential oncogene that inhibits apoptosis. Genes Dev ,1999,13: 2207-2217
[12] Hjiantoniou E, Iseki S, Uney J B, et al. DNazyme-mediated cleavage of Twist transcripts and increase in cellular apoptosis. Biochem Biophys Res Commun, 2003,300:178-181
[13] Shook D, and R Keller. Mechanisms, mechanics and function of epithelial -mesenchymal transitions in early development. Mech Dev, 2003, 120:1351-1383
[14] Hay E D. The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it. Dev Dyn, 2005,233:706-720
[15] Janda E, Lehmann K, Killisch I, et al. Ras and TGF[β]cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol, 2002,156:299-313
[16] Blanco M J, MorenoBueno G, Sarrio D, et al.Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene, 2002,21:3241-3246
[17] Hajra K M, Chen D Y, Fearon E R. The Slug zinc-fingerprotein represses E-cadherin in breast cancer. Cancer Res, 2002,62:1613-1618
[18] Rosivatz E, Becker I, Specht K, et al. Differential expression of the epithelial-mesenchymal transitionregulators snail, SIP1, and twist in gastric cancer. Am J Pathol, 2002,161:1881-1891
[19] Martin T A, Goyal A, Watkins G, et al. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast cancer. Ann Surg Oncol, 2005,12:488-496
[20] Brabletz T, Jung A, Hermann K, et al. Nuclear overexpression of the oncoprotein h-catenin in colorectal cancer is localized predominantly at the invasion front. Pathol Res Pract, 1998,194:701-704
[21] Castanon I, Baylies M K. A Twist in fate: evolutionary comparison of Twist structure and function. Gene, 2002,287(3): 11-22
[22] Comijn J, Berx G, Vermassen P, et al.The two-handed E-box binding zinc finger protein SEP1 downregulates E-cadherin and induces invasion. Mol Cell, 2001,7(6):1267-1278
[23] Thiery J P. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002 ,2(6):442-454
[24] Vega S, Morales A V, Ocana O H, et al. Snail blocks the cell cycle and confers resistance to cell death.Genes Dev, 2004,18(10): 1131-1143
[25] Fujita N, Jaye D L, Kajita M, et al. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell, 2003, 113(2):207-19
[26] Yamashita S, Miyagi C, Fukada T, et al. Zinc transporter LIVI controls epithelial-mesenchymal transition in zebrafish gastrula organizer. Nature, 2004, 429(6989):298-302
[27] Huber M A, Azoitei N, Baumann B, et al. NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest, 2004,114(4):569-581
[28] Vega S, Morales A V, Ocana O H, et al. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev, 2004,18(10): 1131 -1143
[29] Bernards R, Weinberg RA. A progression puzzle. Nature, 2002,418(6900):823
[30] Rosivatz E, Becker I, Specht K, et al. Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol, 2002 ,161(5):1881-1891
[31] Ramaswamy S, Ross K N, Lander E S, et al. A molecular signature of metastasis in primary solid tumors. Nat Genet, 2003, 33(1):49-54
[32] Marsicano G, Wotjak C T, Azad S C, et al. The endogenous cannabinoid system controls extinction of aversive memories. Nature, 2002,418(6897):530-534
[33] Petersen OW, Nielsen HL, Gudjonsson T, et al. Epithelial to mesenchymal transition in human breast cancer can provide a nonmalignant stroma. Am J Pathol, 2003,162(2):391-402
[2] Ries LA G, Eisner M P, Kosary C L, et al. SEER Cancer Statistics Review, 1975-2000, National Cancer Institute.
[3] Lieberman M D, Franseschi D, Marsan B, et al. Esophageal carcinoma: the unusual variants. J Thorac Cardiovasc Surg, 1994,108:1138-1146
[4] Lam A K. Molecular biology of esophageal squamous cell carcinoma. Crit Rev Oncol Hematol, 2000,33(2):71-90
[5] Blot W J. Esophageal cancer trends and risk factors. Semin Oncol, 1994,21: 403-410
[6] Marger R S, Marger D. Carcinoma of the esophagus and tylosis: a lethal genetic combination. Cancer, 1993,72:17-19
[7] Nakakubo Y, Hida Y, Miyamoto M, et al. The prognostic significance of RCAS1 expression in squamous cell carcinoma of the oesophagus. Cancer Lett, 2002,177(1):101-105
[8] Ando N, Ozawa S, Kitagawa Y, et al. Improvement in the results of surgical treatment of advanced squamous esophageal carcinoma during 15 consecutive years. Ann Surg, 2000,232(2):225-232
[9] Adham M, Baulieux J, Mornex F, et al. Combined chemotherapy and radiotherapy followed by surgery in the treatment of patients with squamous cell carcinoma of the esophagus. Cancer, 2000, 89(10): 2144
[10] Sporn M B. The war on cancer. Lancet, 1996,347:1377-1381
[11] Karreth F, Tuveson D A. Twist induces an Epithelial-Mesenchymal transition to facilitate tumor metastasis, Cancer Biol Ther, 2004, 3(ll):1058-1059
[12] Christofori G. Changing neighbours, changing behaviour: cell adhesion molecule-mediated signalling during tumour progression. EMBO J, 2003, 22:2318-2323
[13] Chambers A F, Matrisian L M. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst, 1997, 89:1260-1270
[14] Janda E, Lehmann K, Killisch I, et al. Ras and TGFb cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol, 2002,156:299-313
[15] Thiery J P. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol, 2003,15:740-746
[16] Timmerman L A, Grego-Bessa J, Raya A, et al. Notchpromotes epithelial--mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev, 2004,18:99-115
[17] Lu Z, Ghosh S, Wang Z, et al. Down regulation of caveolin-1 function by EGF leadsto the loss of E-cadherin, increased transcriptional activity of b-catenin, and enhanced tumor cell invasion. Cancer Cell, 2003,4:499-515
[18] Bolos V, Peinado H, Perez-Moreno M A, et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci, 2003, 116: 499-511
[19] Yang J, Mani S A, Donaher J L, et al. Twist, a masterregulator of morphogenesis, plays an essential role intumor metastasis. Cell, 2004,117:927-939
[20] Lee M S, Lowe G N, Strong D D, et al. TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage. J Cell Biochem, 1999,75(4):566-577
[21] Li L, Cserjesi P, Olson E N. Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis. Dev Biol, 1995,172:280-292
[22] Sosic D, Richardson J A, Yu K, et al. Twist regulates cytokine gene expression through a negative feedback loop that represses NF-B activity. Cell, 2003,112: 169-180
[23] Hoek K, Rimm D L, Williams K R, et al. Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas. Cancer Res, 2004,64: 5270-5282
[24] Wang X, Ling M T, Guan X Y, et al. Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells. Oncogene, 2004,23: 474-482
[25] Kwok W K, Ling M T, Lee T W, et al. Up-regulation of TWIST in prostate cancer and its implication as a therapeutic target. Cancer Res, 2005,65: 5153-5162
[26] Dupont J, Fernandez A M, Glackin C A, et al. Insulinlike growth factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor. J Biol Chem, 2001,276:26699-26707
[27] Puisieux A, Valsesia-Wittmann S, Ansieau S, et al. A twist for survival and cancer progression. Br J Cancer, 2006,94(1): 13-17
[28] Kawana K Y, Kawana H, Yoshikawa Y, et al. Nasal immunization of mice with peptide having a cross-neutralization epitope on minor capsid protein L2 of human papillomavirus type 16 elicit (sic) systemic and mucosal antibodies. Vaccine, 2001,19:1496-1502
[29] Niu R F, Zhang L, Xi G M, et al. Up-regulation of Twist induces angiogenesis and correlates with metastasis in hepatocellular carcinoma. J Exp Clin Cancer Res,2007,26(3):385-394
[30] Yan Q Z, Xue Y G, Shuang H, et al. Expression and significance of TWIST basic helix-loop-helix protein over-expression in gastric cancer. Pathology, 2007 39(5):470-475
[31] Yuen H F, Chua C W, Chan Y P, et al. Significance of TWIST and E-cadherin expression in the metastatic progression of prostatic cancer. Histopathology, 2007,50(5):648-658
[32] Ohuchida K, Mizumoto K, Ohhashi S, et al. Twist, a novel oncogene, is upregulated in pancreatic cancer: clinical implication of Twist expression in pancreatic juice. Int J Cancer, 2007,120(8):1634-1640
[33] Sinicrope F A, Ruan S B, Cleary K R, et al. bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis. Cancer Res, 55:237-241
[34] Olek A, Oswald J, Walter J. A modified and improved method for bisulphate based cytosine methylation analysis. Nucleic Acids Res,1996,15(24):5064-5066
[35] Gripp K W, Zackai E H, Stolle C A. Mutations in the human TWIST gene. Hum Mutat, 2000;15(2):150-155
[36] Jan Y N, Jan L Y. Functional gene cassettes in development. Proc Natl Acad Sci USA, 1993,90: 8305-8307
[37] Thisse B, Stoetzel C, Messal M, et al. The twist gene:isolation of a Drosophila zygotic gene necessary for the establishment of dorso-ventral pattern. Nucleic Acids Res, 1987,15:3439-3453
[38] Park R E, Kosman D, Bier E, et al. The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev, 1992,6:1728-1739
[39] Leptin M. twist and snail as positive and negative regulators duringDrosophila mesoderm development. Genes Dev, 1991,5:1568-1576
[40] Maestro R, Dei Tos A P, Hamamori Y, et al. Twist is a potential oncogene that inhibits apoptosis. Genes Dev, 1999,13:2207-2217
[41] Rosivatz E, Becker I, Specht K, et al. Differential expression of the epithelial -mesenchymal transition regulators Snail, SIP1, and Twist in gastric cancer. Am J Pathol, 2002,161:1881-1891
[42] Martin T A, Goyal A, Watkins G, et al. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast cancer. Ann Surg Oncol, 2005,12(6):488-496
[43] Hoek K, Rimm D L, Williams K R, et al. Expression profiling reveals novel pathways in the transformation of melanocytes to melanomas. Cancer Res, 2004 64(15):5270-5282
[44] Song L B, Liao W T, Mai H Q, et al. The clinical significance of twist expression in nasopharyngeal carcinoma. Cancer Lett, 2006,242(2):258-265
[45] Yuen H F, Chan Y P, Wong M L, et al. Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis. J Clin Pathol, 2007,60(5):510-514
[46] Furlong E E, Andersen E C, Null. B, et al. Patterns of gene expression during Drosophila mesoderm development. Science, 2001,293:1629-1633
[47] Chen Z F, Behringer R R. Twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev, 1995,9:686-699
[48] Soo K, O'Rourke M P, Khoo P L, et al. Twist function is required for the morphogenesis of the cephalic neural tube and the differentiation of the cranial neural crest cells in the mouse embryo. Dev Biol, 2002,247:251-270
[49] Meulemans D, Bronner-Fraser M. Gene-regulatory interactions in neural crest evolution and development. Dev Cell, 2004,7:291-299
[50] Maestro R, Dei Tos A P, Hamamori Y, et al. TWIST is a potential oncogene that inhibits apoptosis. Genes Dev, 1999,13:2207-2217
[51] Valsesia-Wittmann S, Magdeleine M, Dupasquier S, et al. Oncogenic cooperation between H-TWIST and the N-Myc overrides failsafe programs in cancer cells. Cancer Cell, 2004,6:625-630
[52] Sosic D, Richardson J A, Yu K, et al. TWIST regulates cytokine gene expression through a negative feedback loop that represses NF-kappa B activity. Cell, 2003,112:169-180
[53] Janda E, Lehmann K, Killisch I, et al. Ras and TGF[h] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol, 2002,156:299-313
[54] Batlle E, Sancho E, Franci C, et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol, 2000, 2:84-89
[55] Cano A, Perez-Moreno M A, Rodrigo I, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.Nat Cell Biol, 2000,2:76-83
[56] Hajra K M, Chen D Y, Fearon E R. The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res, 2002,62:1613-1618
[57] Comijn J, Berx G, Vermassen P, et al. The twohanded E-box binding zinc finger protein SIP1downregulates E-cadherin and induces invasion. Mol Cell, 2001, 7:1267-1278
[58] Shiren S, Xiao X N, Yan Q Z, et al. Hypoxia-inducible factor-1a induces Twist expression in tubular epithelial cells subjected to hypoxia, leading to epithelial-to-mesenchymal transition. Kidney Int, 2009,March, 11:1-10
[1] Yang J, Mani S A, Donaher J L, et al. Twist, a masterregulator of morphogenesis, plays an essential role intumor metastasis. Cell, 2004,117:927-939
[2] Thisse B, Stoetzel C, El Messal M, et al. The twist gene: Isolation of a Drosophila zygotic gene necessary for the establishment of dorso-ventral pattern. Nucleic Acids Res, 1987,15:3439-3453
[3] Jan Y N, Jan L Y. Functional gene cassettes in development. Proc Natl Acad Sci USA, 1993,90: 8305- 8307
[4] Ip Y T, Park R E, Kosman D, et al. The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev,1992, 6: 1728-1739
[5] Leptin M. Twist and Snail as positive and negative r egulators during Drosophilamesoderm development. Genes Dev, 1991,5:1568-1576
[6] Li L, Cserjesi P, Olson E N. Dermo-1: a novel twist-related bHLH protein expressed in the developing dermis. Dev Biol ,1995,172:280-292
[7] Bialek P, Kern B, Yang X, et al. A twist code determines the onset of osteoblast differentiation. Dev Cell, 2004, 6: 423-435
[8] Sosic D, Richardson J A, Yu K, et al. Twist regulates cytokine gene expression through a negative feedback loop that represses NF-B activity. Cell, 2003,112: 169-180
[9] Pantke O A, Cohen Jr MM, Witkop Jr CJ, et al.The Saethre-Chotzen syndrome. Birth Defects OrigArti Ser,1975,11:190-225
[10] Chen Z F, Behringer R R. Twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev, 1995,9: 686-699
[11] Maestro R, Dei Tos AP, Hamamori Y, et al. Twist is a potential oncogene that inhibits apoptosis. Genes Dev ,1999,13: 2207-2217
[12] Hjiantoniou E, Iseki S, Uney J B, et al. DNazyme-mediated cleavage of Twist transcripts and increase in cellular apoptosis. Biochem Biophys Res Commun, 2003,300:178-181
[13] Shook D, and R Keller. Mechanisms, mechanics and function of epithelial -mesenchymal transitions in early development. Mech Dev, 2003, 120:1351-1383
[14] Hay E D. The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it. Dev Dyn, 2005,233:706-720
[15] Janda E, Lehmann K, Killisch I, et al. Ras and TGF[β]cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J Cell Biol, 2002,156:299-313
[16] Blanco M J, MorenoBueno G, Sarrio D, et al.Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene, 2002,21:3241-3246
[17] Hajra K M, Chen D Y, Fearon E R. The Slug zinc-fingerprotein represses E-cadherin in breast cancer. Cancer Res, 2002,62:1613-1618
[18] Rosivatz E, Becker I, Specht K, et al. Differential expression of the epithelial-mesenchymal transitionregulators snail, SIP1, and twist in gastric cancer. Am J Pathol, 2002,161:1881-1891
[19] Martin T A, Goyal A, Watkins G, et al. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast cancer. Ann Surg Oncol, 2005,12:488-496
[20] Brabletz T, Jung A, Hermann K, et al. Nuclear overexpression of the oncoprotein h-catenin in colorectal cancer is localized predominantly at the invasion front. Pathol Res Pract, 1998,194:701-704
[21] Castanon I, Baylies M K. A Twist in fate: evolutionary comparison of Twist structure and function. Gene, 2002,287(3): 11-22
[22] Comijn J, Berx G, Vermassen P, et al.The two-handed E-box binding zinc finger protein SEP1 downregulates E-cadherin and induces invasion. Mol Cell, 2001,7(6):1267-1278
[23] Thiery J P. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002 ,2(6):442-454
[24] Vega S, Morales A V, Ocana O H, et al. Snail blocks the cell cycle and confers resistance to cell death.Genes Dev, 2004,18(10): 1131-1143
[25] Fujita N, Jaye D L, Kajita M, et al. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell, 2003, 113(2):207-19
[26] Yamashita S, Miyagi C, Fukada T, et al. Zinc transporter LIVI controls epithelial-mesenchymal transition in zebrafish gastrula organizer. Nature, 2004, 429(6989):298-302
[27] Huber M A, Azoitei N, Baumann B, et al. NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest, 2004,114(4):569-581
[28] Vega S, Morales A V, Ocana O H, et al. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev, 2004,18(10): 1131 -1143
[29] Bernards R, Weinberg RA. A progression puzzle. Nature, 2002,418(6900):823
[30] Rosivatz E, Becker I, Specht K, et al. Differential expression of the epithelial-mesenchymal transition regulators snail, SIP1, and twist in gastric cancer. Am J Pathol, 2002 ,161(5):1881-1891
[31] Ramaswamy S, Ross K N, Lander E S, et al. A molecular signature of metastasis in primary solid tumors. Nat Genet, 2003, 33(1):49-54
[32] Marsicano G, Wotjak C T, Azad S C, et al. The endogenous cannabinoid system controls extinction of aversive memories. Nature, 2002,418(6897):530-534
[33] Petersen OW, Nielsen HL, Gudjonsson T, et al. Epithelial to mesenchymal transition in human breast cancer can provide a nonmalignant stroma. Am J Pathol, 2003,162(2):391-402