Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells
Jian-Jun Xie
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorCorresponding Author
Li-Yan Xu
Department of Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou, People's Republic of China
Fax: +86-754-88900847
Li-Yan Xu, Institute of Oncologic Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou 515041, People's Republic of China
En-Min Li, Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou 515041, People's Republic of China
Search for more papers by this authorYang-Min Xie
Department of Experimental Animal Center, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorHai-Hua Zhang
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorWei-Jia Cai
Department of Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorFei Zhou
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorZhong-Ying Shen
Department of Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorCorresponding Author
En-Min Li
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Fax: +86-754-88900847
Li-Yan Xu, Institute of Oncologic Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou 515041, People's Republic of China
En-Min Li, Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou 515041, People's Republic of China
Search for more papers by this authorJian-Jun Xie
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorCorresponding Author
Li-Yan Xu
Department of Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou, People's Republic of China
Fax: +86-754-88900847
Li-Yan Xu, Institute of Oncologic Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou 515041, People's Republic of China
En-Min Li, Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou 515041, People's Republic of China
Search for more papers by this authorYang-Min Xie
Department of Experimental Animal Center, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorHai-Hua Zhang
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorWei-Jia Cai
Department of Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorFei Zhou
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorZhong-Ying Shen
Department of Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou, People's Republic of China
Search for more papers by this authorCorresponding Author
En-Min Li
Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou, People's Republic of China
Fax: +86-754-88900847
Li-Yan Xu, Institute of Oncologic Pathology, The Key Immunopathology Laboratory of Guangdong Province, Medical College of Shantou University, Shantou 515041, People's Republic of China
En-Min Li, Department of Biochemistry and Molecular Biology, Medical College of Shantou University, Shantou 515041, People's Republic of China
Search for more papers by this authorAbstract
Ezrin, which crosslinks the cytoskeleton and plasma membrane, is involved in the growth and metastatic potential of cancer cells. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its roles and the underlying mechanism(s) remain unclear. In our study, we first showed that ezrin in ESCC cell is expressed in the nucleus as well as in the cytoplasm and plasma membrane. Then, by using RNAi, we revealed that interference of ezrin expression suppressed the growth, adhesion and invasiveness of ESCC cells. Tumorigenesis experiments revealed that ezrin may directly regulate tumor formation in vivo. To explore the molecular mechanisms through which ezrin contributes to the proliferation and invasiveness of ESCC cells, we used cDNA microarrays to analyze ezrin knockdown cells and the control cells; of 39,000 genes examined, 297 were differentially expressed upon ezrin knockdown, including some proliferation- and invasiveness-related genes such as ATF3, CTGF and CYR61. Furthermore, pathway analysis showed that ezrin knockdown led to decreased activation of the TGF-β and MAPK pathways, and ezrin-mediated cell invasiveness alteration was dependent on the activation of these pathways. Finally, immunohistochemical staining on 80 ESCC specimens and 50 normal esophageal mucosae revealed that the expression levels of 3 altered genes involved in the regulation of cell proliferation and tumor metastasis, including CTGF, CYR61 and ATF3, were altered in ESCCs, and their expression pattern correlated with ezrin expression. Taken together, we propose that ezrin might function in the growth and invasiveness of ESCC cells through the MAPK and TGF-β pathways. © 2008 Wiley-Liss, Inc.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
| Filename | Description |
|---|---|
| IJC_24216_sm_SuppTab1-2.doc69 KB | Supplementary table 1 Antibodies used in these studies. Supplementary table 2 Primer sequences for RT-PCR |
| IJC_24216_sm_SuppFig1-3.doc338 KB | Supplementary figure 1 Ezrin antibody (sc-58758, Santa Cruz) was used for immunofluorescence analysis of ezrin expression in EC109 cells and SHEE cells. Representative images showing localization of ezrin (green stain) and staining of DAPI (blue stain). Immunofluorescence with the secondary antibody alone was using as blank control. Bar, 20μm. Supplementary figure 2 Quantitative expressions of certain differentially expressed genes were detected by using RT-PCR (A) or western blotting (B). Signal intensities of the bands were quantified by densitometric scanning, normalized by internal control (GAPDH for RT-PCR and β-actin for western blotting) and relative to the expression of corresponding gene in EC109 cells. Supplementary figure 3 Quantitative expressions of ezrin, CTGF, CYR61, SDC2 and ATF3 in the ezrin re-expression cells were determined by using RT-PCR. Signal intensities of the bands were quantified by densitometric scanning, normalized by internal control (GAPDH) and relative to the expression of corresponding gene in EC109 cells. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1 Isono K,Sato H,Nakayama K. Results of a nationwide study on the three-field lymph node dissection of esophageal cancer. Oncology 1991; 48: 411–20.
- 2
Aznavoorian S,Murphy AN,Stetler-Stevenson WG,Liotta LA.
Molecular aspects of tumor cell invasion and metastasis.
Cancer
1993;
71:
1368–83.
10.1002/1097-0142(19930215)71:4<1368::AID-CNCR2820710432>3.0.CO;2-L CAS PubMed Web of Science® Google Scholar
- 3
Pisani P,Parkin DM,Bray F,Ferlay J.
Estimates of the worldwide mortality from 25 cancers in 1990.
Int J Cancer
1999;
83:
18–29.
10.1002/(SICI)1097-0215(19990924)83:1<18::AID-IJC5>3.0.CO;2-M CAS PubMed Web of Science® Google Scholar
- 4 Toh Y,Kuwano H,Mori M,Nicolson GL,Sugimachi K. Overexpression of metastasis-associated MTA1 mRNA in invasive oesophageal carcinomas. Br J Cancer 1999; 79: 1723–26.
- 5 Xie JJ,Xu LY,Zhang HH,Cai WJ,Mai RQ,Xie YM,Yang ZM,Niu YD,Shen ZY,Li EM. Role of fascin in the proliferation and invasiveness of esophageal carcinoma cells. Biochem Biophys Res Commun 2005; 337: 355–62.
- 6 Zhang H,Xu L,Xiao D,Xie J,Zeng H,Cai W,Niu Y,Yang Z,Shen Z,Li E. Fascin is a potential biomarker for early-stage oesophageal squamous cell carcinoma. J Clin Pathol 2006; 59: 958–64.
- 7 Vaheri A,Carpén O,Heiska L,Helander TS,Jääskeläinen J,Majander-Nordenswan P,Sainio M,Timonen T,Turunen O. The ezrin protein family: membrane-cytoskeleton interactions and disease associations. Curr Opin Cell Biol 1997; 9: 659–66.
- 8 Kishore R,Qin G,Luedemann C,Bord E,Hanley A,Silver M,Gavin M,Yoon YS,Goukassian D,Losordo DW. The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-α-induced transcriptional repression of cyclin A. J Clin Invest 2005; 115: 1785–96.
- 9 Batchelor CL,Woodward AM,Crouch DH. Nuclear ERM (ezrin, radixin, moesin) proteins: regulation by cell density and nuclear import. Exp Cell Res 2004; 296: 208–22.
- 10 Böhling T,Turunen O,Jääskeläinen J,Carpen O,Sainio M,Wahlström T,Vaheri A,Haltia M. Ezrin expression in stromal cells of capillary hemangioblastoma. An immunohistochemical survey of brain tumors. Am J Pathol 1996; 148: 367–73.
- 11 Elliott BE,Meens JA,SenGupta SK,Louvard D,Arpin M. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res 2005; 7: R365–R373.
- 12 Khanna C,Wan X,Bose S,Cassaday R,Olomu O,Mendoza A,Yeung C,Gorlick R,Hewitt SM,Helman LJ. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med 2004; 10: 182–6.
- 13 Yu Y,Khan J,Khanna C,Helman L,Meltzer PS,Merlino G. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med 2004; 10: 175–81.
- 14 Kaul SC,Mitsui Y,Komatsu Y,Reddel RR,Wadhwa R. A highly expressed 81-kDa protein in immortalized mouse fibroblasts: its proliferative function and identity with ezrin. Oncogene 1996; 13: 1231–7.
- 15 Bretscher A. Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J Cell Biol 1989; 108: 921–31.
- 16 Shen ZY,Xu LY,Li EM,Li JT,Chen MH,Shen J,Zeng Y,Shen ZY,Xu LY,Li EM. Ezrin, actin and cytoskeleton in apoptosis of esophageal epithelial cells induced by arsenic trioxide. Int J Mol Med 2003; 12: 341–7.
- 17 Zeng H,Xu L,Xiao D,Zhang H,Wu X,Zheng R,Li Q,Niu Y,Shen Z,Li E. Altered expression of ezrin in esophageal squamous cell carcinoma. J Histochem Cytochem 2006; 54: 889–96.
- 18 Shen ZY,Xu LY,Li EM,Cai WJ,Shen J,Chen MH,Cen S,Tsao SW,Zeng Y. The multistage process of carcinogenesis in human esophageal epithelial cells induced by human papillomavirus. Oncol Rep 2004; 11: 647–54.
- 19 Mosmann TR,Bond MW,Coffman RL,Ohara J,Paul WE. T-cell and mast cell lines respond to B-cell stimulatory factor 1. Proc Natl Acad Sci USA 1986; 83: 5654–8.
- 20 Kim D,Kim S,Koh H,Yoon SO,Chung AS,Cho KS,Chung J. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J 2001; 15: 1953–62.
- 21 Iannetti A,Pacifico F,Acquaviva R,Lavorgna A,Crescenzi E,Vascotto C,Tell G,Salzano AM,Scaloni A,Vuttariello E,Chiappetta G,Formisano S, et al. The neutrophil gelatinase-associated lipocalin (NGAL), a NF-κB-regulated gene, is a survival factor for thyroid neoplastic cells. Proc Natl Acad Sci USA 2008; 105: 14058–63.
- 22 Malinda KM,Nomizu M,Chung M,Delgado M,Kuratomi Y,Yamada Y,Kleinman HK,Ponce ML. Identification of laminin α1 and β1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting. FASEB J 1999; 13: 53–62.
- 23 Berryman M,Franck Z,Bretscher A. Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci 1993; 105: 1025–43.
- 24 Brigstock DR. The CCN family: a new stimulus package. J Endocrinol 2003; 178: 169–75.
- 25 Grotendorst GR,Okochi H,Hayashi N. A novel transforming growth factor β response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ 1996; 7: 469–80.
- 26 Ranganathan P,Agrawal A,Bhushan R,Chavalmane AK,Kalathur RK,Takahashi T,Kondaiah P. Expression profiling of genes regulated by TGF-β: differential regulation in normal and tumour cells. BMC Genomics 2007; 8: 98.
- 27 Leivonen SK,Kähäri VM. Transforming growth factor-β signaling in cancer invasion and metastasis. Int J Cancer 2007; 121: 2119–24.
- 28 Zhang Y,Hu MY,Wu WZ,Wang ZJ,Zhou K,Zha XL,Liu KD. The membrane-cytoskeleton organizer ezrin is necessary for hepatocellular carcinoma cell growth and invasiveness. J Cancer Res Clin Oncol 2006; 132: 685–97.
- 29 Parlato S,Giammarioli AM,Logozzi M,Lozupone F,Matarrese P,Luciani F,Falchi M,Malorni W,Fais S. CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway. EMBO J 2000; 19: 5123–34.
- 30 Wang LD,Zhou Q,Yang WC,Yang CS. Apoptosis and cell proliferation in esophageal precancerous and cancerous lesions: study of a high-risk population in northern China. Anticancer Res 1999; 19: 369–74.
- 31 Makitie T,Carpen O,Vaheri A,Kivela T. Ezrin as a prognostic indicator and its relationship to tumor characteristics in uveal malignant melanoma. Invest Ophthalmol Vis Sci 2001; 42: 2442–9.
- 32 Akisawa N,Nishimori I,Iwamura T,Onishi S,Hollingsworth MA. High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential. Biochem Biophys Res Commun 1999; 258: 395–400.
- 33 Hunter KW. Ezrin, a key component in tumor metastasis. Trends Mol Med 2004; 10: 201–4.
- 34 Takeuchi K,Sato N,Kasahara H,Funayama N,Nagafuchi A,Yonemura S,Tsukita S,Tsukita S. Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol 1994; 125: 1371–84.
- 35 Liotta LA,Tryggvason K,Garbisa S,Hart I,Foltz CM,Shafie S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 1980; 284: 67–8.
- 36 Koliopanos A,Friess H,di Mola FF,Tang WH,Kubulus D,Brigstock D,Zimmermann A,Büchler MW. Connective tissue growth factor gene expression alters tumor progression in esophageal cancer. World J Surg 2002; 26: 420–7.
- 37 Di Martino E,Wild CP,Rotimi O,Darnton JS,Olliver RJ,Hardie LJ. IGFBP-3 IGFBP-10 (CYR61) up-regulation during the development of Barrett's oesophagus and associated oesophageal adenocarcinoma: potential biomarkers of disease risk. Biomarkers 2006; 11: 547–61.
- 38 Granés F,Berndt C,Roy C,Mangeat P,Reina M,Vilaró S. Identification of a novel Ezrin-binding site in syndecan-2 cytoplasmic domain. FEBS Lett 2003; 547: 212–16.
- 39 Chen L,Klass C,Woods A. Syndecan-2 regulates transforming growth factor-β signaling. J Biol Chem 2004; 279: 15715–18.
Citing Literature
