Hong-Jian Wang, Jin-Shui Zhu, Qiang Zhang, Qun Sun, Hua Guo, Department of Gastroenterology, The Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200233, China
Author contributions: Wang HJ wrote the paper; Zhu JS designed the research; Wang HJ and Guo H performed the research; Zhang Q and Sun Q provided new reagent and analytic tools and analyzed data.
Supported by Natural Science Foundation of Shanghai, No. 04ZB14072
Correspondence to: Jin-Shui Zhu, Professor, Department of Gastroenterology, The Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200233, China. firstname.lastname@example.org
Telephone: +86-21-64369181 Fax: +86-21-64837019
Received: December 16, 2008 Revised: March 2, 2009
Accepted: March 9, 2009
Published online: April 28, 2009
AIM: To investigate the ezrin expression in normal colorectal mucosa and colorectal cancer tissues, and study the correlation between ezrin expression in colorectal cancer tissues and tumor invasion and metastasis.
METHODS: Eighty paraffin-embedded cancer tissue samples were selected from primary colorectal adenocarcinoma. Twenty-eight patients had well-differentiated, 22 had moderately differentiated and 30 had poorly differentiated adenocarcinoma. Forty-five patients and 35 patients had lymph node metastasis. Forty-five patients were of Dukes A to B stage, and 35 were of C to D stage. Another 22 paraffin-embedded tissue blocks of normal colorectal epithelium (> 5 cm away from the edge of the tumor) were selected as the control group. All patients with colorectal cancer were treated surgically and diagnosed histologically, without preoperative chemotherapy or radiotherapy. The immunohistochemistry was used to detect the ezrin expression in paraffin-embedded normal colorectal mucosa tissues and colorectal cancer tissue samples.
RESULTS: Ezrin expression in colorectal cancer was significantly higher than in normal colorectal mucosa (75.00% vs 9.09%, P < 0.01), and there was a close relationship between ezrin expression and the degree of tumor differentiation, lymph node metastasis and Dukes stage (88.46% vs 50.00%, P < 0.01; 94.28% vs 51.11%, P < 0.01; 94.28% vs 51.11%, P < 0.01).
CONCLUSION: Ezrin expression is obviously higher in colorectal cancer tissues than in normal colorectal mucosa tissues, and the high level of ezrin expression is closely related to the colorectal cancer invasion and metastasis process.
© 2009 The WJG Press and Baishideng. All rights reserved.
Key words: Colorectal cancer; Ezrin; Malignant tumor; Invasion; Metastasis; Immunohistochemistry
Peer reviewers: Alain L Servin, PhD, Faculty of Pharmacy, French National Institute of Health and Medical Research, Unit 756, Rue J.-B. Clément, F-922296 Châtenay-Malabry, France; Filip Braet, Associate Professor, Australian Key Centre for Microscopy and Microanalysis, Madsen Building (F09), The University of Sydney, Sydney NSW 2006, Australia
Wang HJ, Zhu JS, Zhang Q, Sun Q, Guo H. High level of ezrin expression in colorectal cancer tissues is closely related to tumor malignancy. World J Gastroenterol 2009; 15(16): 2016-2019 Available from: URL: http://www.wjgnet.com/1007-9327/15/2016.asp DOI: http://dx.doi.org/10.3748/wjg.15.2016
Ezrin belongs to the ezrin/radixin/moesin (ERM) protein family, which act as membrane organizers and linkers between the plasma membrane and cytoskeleton[1,2]. Ezrin is mainly expressed on the cell surface to maintain the polarity of endothelial cells. Recent studies have found that, through regulating adhesion molecules and signal transduction pathways, ezrin is involved in cell-cell and cell-matrix interactions, and might play an important role in the process of tumor cell invasion and metastasis. Overexpression of ezrin protein is correlated with the metastatic potential of several cancers[5-8], and a high level of ezrin protein expression can induce conversion of a variety of cell lines, as well as abnormal hyperplasia. Tumor cell lines with stronger metastatic abilities are usually accompanied by overexpression of ezrin. Through testing the expression of ezrin protein in normal colonic mucosa and colorectal cancer tissues, we aimed to establish the relationship between ezrin expression and clinical parameters, evaluate its molecular action mechanisms in the process of colorectal cancer carcinogenesis, invasion and metastasis, and provide the evidence for clinical prognosis and suitable adjuvant therapy.
MATERIALS AND METHODS
Patients and their pathological samples
The immunohistochemistry was performed in paraffin-embedded tissue samples. Eighty colorectal adenocarcinoma patients diagnosed by postoperative pathology were investigated. There were 44 male and 36 female patients, whose ages ranged from 31 to 80 years, with an average age of 55.5 years. Histologically, 28 patients had well-differentiated, 22 had moderately differentiated, and 30 had poorly differentiated adenocarcinoma. Forty-five patients were without and 35 patients had lymph node metastasis. Forty-five patients were of Dukes A to B stage, and 35 were of C to D stage. Another 22 paraffin-embedded tissue blocks of normal colorectal epithelium (> 5 cm away from the edge of the tumor) was the control group.
Drugs and reagents
Mouse anti-human ezrin mAb was purchased from Fujian Maixin Biotechnology Development Co. Ltd, and SP kit DAB from Beijing Zhong Shan Jinqiao Biotechnology Development Co. Ltd. Experiments were performed following the instructions of the manufacturers. PBS (0.01 mmol/L) was used to replace the first antibody as a negative control, while the normal colorectal mucosa was a positive control.
Each stained slide was assessed and given a score according to the classification standard of Mathew et al: score 0, no expression; score 1, < 50% of cells staining positive expression or less; score 2, ≥ 50% of cells staining positive expression. Score 0-1 was recorded as negative, and score 2 recorded as positive.
SPSS for Windows version 11.0 was used for statistical analyses. The c2 test was used in the analysis of the relationship between ezrin and colorectal cancer clinicopathological parameters. P ≤ 0.05 was considered as a significant difference.
The positive expression of ezrin in colorectal cancer was significantly higher than that in normal colorectal mucosa (Figure 1A-E). The positive rate of ezrin protein in normal colorectal mucosa was 9.09% (2/22) and 75.00% (60/80) in colorectal cancer tissues. There were significant differences between the two groups (75.00% vs 9.09%, P < 0.01), as shown in Table 1.
Relationship between ezrin expression in colorectal cancer tissues and clinicopathological parameters
There was a close relationship between ezrin expression and the degree of tumor differentiation, lymph node metastasis and Dukes stage (88.46% vs 50.00%, P < 0.01; 94.28% vs 51.11%, P < 0.01; 94.28% vs 51.11%, P < 0.01), as shown in Table 2.
Ezrin protein expression in specific cell membrane regions is mainly involved in the connection between the epithelial cell cytoskeleton and the cell membrane, through membrane surface signaling molecules and some transmembrane signal transduction pathway. It participates in the regulation of cell survival, adhesion, proliferation and migration processes. Recent studies have found that ezrin protein may play an important role in the tumorigenesis, development, invasion and metastasis process, probably through regulating adhesion molecules and participating in cell signal transduction, and other channels in the tumor[12-17]. Ezrin protein is an indispensable factor for tumor cell metastasis of osteosarcoma, breast cancer, nasopharyngeal carcinoma, and prostate cancer. In addition, in malignant tumor tissues, there are also changes in subcellular localization of ezrin expression. Moilanen et al found that ezrin expression in normal ovarian epithelial cells is a kind of cell polarity expression, and that ezrin expression in malignant ovarian tumor cells is more diffusive, with a different degree of tumor cell differentiation, and the location and intensity of ezrin expression in cells is quite different. Therefore, we speculate that ezrin subcellular localization in normal cells forms the foundation of various physiological functions and cell structure. Abnormal ezrin expression or distribution will also lead to abnormal cell structure and physiological function, and accordingly, these abnormal changes participate in the occurrence, development, invasion and metastasis of malignant tumors.
The role of ezrin in tumor progression is very important and deserves much attention. Recent studies have found that ezrin is a key factor in Fas-mediated apoptosis, in the P-gp1-mediated multidrug resistance of cancers, and in cannibalism of metastatic tumors. The active ezrin C-terminal is connected with the actin cytoskeleton, and the N-terminal is connected with cell adhesion molecules such as E-cadherin, and CD44[25,26], etc. Ezrin participates in regulating cell-cell and cell-extracellular matrix adhesion, thus influencing tumor cell invasion and other biological behavior[27-30]. CD44 is a cellular membrane receptor which can specially recognize hyaluronic acid and collagen, and regulate cell-cell and cell-extracellular matrix adhesion. Some studies have found that ezrin, CD44 and CD44 variants could make up a compound that is co-expressed in the tumor cells. Pujuguet et al have found that ezrin can regulate E-cadherin expression in the cell membrane through Rho protein, thereby regulating cell adhesion. At the same time, ezrin also has regulating function in the E-cadherin membrane localization, and activated ezrin can make the E-cadherin protein aggregate in the cell, thereby undermining the cell-to-cell contact and intercellular adhesive ability, and the overexpression of ezrin in the tissues also has the same function of weakening the intercellular adhesion. Through activation of RhoA and the MAPK pathway, ezrin can promote the cell adhesion plaque formation, thereby promoting the adhesive function between the tumor cells and other cells, as well as stoma cells. Therefore, we believe, through participation in the formation of the cell adhesion plaque, cytoskeletal connections and cell surface compounds assembly, and other biological functions, ezrin protein mediates and regulates cell-cell and cell-extracellular matrix adhesion, and is also involved in the malignant tumor invasion and metastasis process. This study showed that, the overexpression of ezrin in colorectal cancer tissues may be involved in cancer invasion and metastasis. The studies on the correlation between ezrin protein and cancer might help us further reveal the tumor invasion and metastasis mechanism, and find the targets for inhibiting tumor metastasis, or indicators that forecasts the prognosis of patients with tumors.
The authors thank Dr. Yun-Hai Dai and Dr. Xiao-Peng Xiong of Department of Nuclear Medicine, Affiliated Renji Hospital of Shanghai Jiao Tong University for their valuable discussions and comments.
Ezrin belongs to the ezrin/radixin/moesin (ERM) protein family, which act as membrane organizers and linkers between the plasma membrane and cytoskeleton. Ample evidence has indicated that ezrin is regarded as a metastatic determinant and a key component in tumor progression and metastasis; however, its role in the process of colorectal cancer growth and metastasis is not clearly understood.
Recent studies have found that a high level of ezrin protein expression can induce a variety of cell line conversions, as well as abnormal hyperplasia. Ezrin is a key factor in Fas-mediated apoptosis, the P-gp1-mediated multidrug resistance of cancers, and cannibalism of metastatic tumors.
This preliminary study about ezrin in colorectal cancer growth and metastasis may pave the way for further clinical studies on colorectal cancer dissemination and metastasis.
Ezrin belongs to the ERM protein family, which are expressed in specific cell membrane regions, and acts as membrane organizers and linkers between the plasma membrane and epithelial cell cytoskeleton.
This study is interesting, and discusses the ezrin expression in normal colorectal mucosa and colorectal cancer tissues, and the clinical relevance of ezrin expression to tumor invasion and metastasis. The study was carefully performed and the data and conclusions drawn are sound.
3 Wald FA, Oriolo AS, Mashukova A, Fregien NL, Langshaw AH, Salas PJ. Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells. J Cell Sci 2008; 121: 644-654 PubMed DOI
5 Koon N, Schneider-Stock R, Sarlomo-Rikala M, Lasota J, Smolkin M, Petroni G, Zaika A, Boltze C, Meyer F, Andersson L, Knuutila S, Miettinen M, El-Rifai W. Molecular targets for tumour progression in gastrointestinal stromal tumours. Gut 2004; 53: 235-240 PubMed DOI
7 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: 182-186 PubMed DOI
8 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: 175-181 PubMed DOI
9 Kaul SC, Mitsui Y, Komatsu Y, Reddel RR, Wadhwa R. A highly expressed 81 kDa protein in immortalized mouse fibroblast: its proliferative function and identity with ezrin. Oncogene 1996; 13: 1231-1237 PubMed
10 Lamb RF, Ozanne BW, Roy C, McGarry L, Stipp C, Mangeat P, Jay DG. Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts. Curr Biol 1997; 7: 682-688 PubMed DOI
12 Khanna C, Khan J, Nguyen P, Prehn J, Caylor J, Yeung C, Trepel J, Meltzer P, Helman L. Metastasis-associated differences in gene expression in a murine model of osteosarcoma. Cancer Res 2001; 61: 3750-3759 PubMed
13 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: 182-186 PubMed DOI
14 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: 175-181 PubMed DOI
15 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 PubMed DOI
16 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 PubMed DOI
18 Ferrari S, Zanella L, Alberghini M, Palmerini E, Staals E, Bacchini P. Prognostic significance of immunohistochemical expression of ezrin in non-metastatic high-grade osteosarcoma. Pediatr Blood Cancer 2008; 50: 752-756 PubMed DOI
19 Li Q, Wu M, Wang H, Xu G, Zhu T, Zhang Y, Liu P, Song A, Gang C, Han Z, Zhou J, Meng L, Lu Y, Wang S, Ma D. Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett 2008; 261: 55-63 PubMed DOI
20 Shen ZH, Chen XY, Chen J. Impact of up-regulating Ezrin expression by Epstein-Barr virus latent membrane protein 1 on metastasis ability of nasopharyngeal carcinoma cells. Ai Zheng 2008; 27: 165-169 PubMed
21 Musiał J, Sporny S, Nowicki A. Prognostic significance of E-cadherin and ezrin immunohistochemical expression in prostate cancer. Pol J Pathol 2007; 58: 235-243 PubMed
24 Lugini L, Matarrese P, Tinari A, Lozupone F, Federici C, Iessi E, Gentile M, Luciani F, Parmiani G, Rivoltini L, Malorni W, Fais S. Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells. Cancer Res 2006; 66: 3629-3638 PubMed DOI
25 Tsukita S, Oishi K, Sato N, Sagara J, Kawai A, Tsukita S. ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 1994; 126: 391-401 PubMed DOI
26 Yonemura S, Hirao M, Doi Y, Takahashi N, Kondo T, Tsukita S, Tsukita S. Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 1998; 140: 885-895 PubMed DOI
33 Birukov KG, Leitinger N, Bochkov VN, Garcia JG. Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins. Microvasc Res 2004; 67: 18-28 PubMed DOI
S- Editor Tian L L- Editors Ma JY and Kerr C E- Editor Zheng XM