Brief Articles
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Apr 28, 2009; 15(16): 2016-2019
Published online Apr 28, 2009. doi: 10.3748/wjg.15.2016
High level of ezrin expression in colorectal cancer tissues is closely related to tumor malignancy
Hong-Jian Wang, Jin-Shui Zhu, Qiang Zhang, Qun Sun, Hua Guo
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.
Correspondence to: Jin-Shui Zhu, Professor, Department of Gastroenterology, The Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200233, China. zhujs1803@hotmail.com
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

Abstract

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.

Key Words: Colorectal cancer, Ezrin, Malignant tumor, Invasion, Metastasis, Immunohistochemistry



INTRODUCTION

Ezrin belongs to the ezrin/radixin/moesin (ERM) protein family, which act as membrane organizers and linkers between the plasma membrane and cytoskeleton[12]. Ezrin is mainly expressed on the cell surface to maintain the polarity of endothelial cells[3]. 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[4]. Overexpression of ezrin protein is correlated with the metastatic potential of several cancers[58], and a high level of ezrin protein expression can induce conversion of a variety of cell lines, as well as abnormal hyperplasia[9]. Tumor cell lines with stronger metastatic abilities are usually accompanied by overexpression of ezrin[10]. 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.

Result judgment

Each stained slide was assessed and given a score according to the classification standard of Mathew et al[11]: 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.

Statistical analyses

SPSS for Windows version 11.0 was used for statistical analyses. The χ2 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.

RESULTS

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.

Table 1 Ezrin expression in different colorectal tissues n (%).
GroupnPositive expression (%)
Normal colorectal mucosa222 (9.09)b
Colorectal cancer tissues8060 (75.00)
Figure 1
Figure 1 Ezrin expression (HE, × 400). A: Normal colorectal mucosa; B: Well-differentiated adenocarcinoma; C: Poorly differentiated adenocarcinoma; D: Adenocarcinoma without lymph node metastasis; E: Adenocarcinoma with lymph node metastasis.
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.

Table 2 Relationship between ezrin expression in colorectal cancer tissues and clinicopathological parameters n (%).
Clinicopathological parametersnEzrin positive expression (%)
Well-differentiated2814 (50.00)b
Moderately and poorly differentiated5246 (88.46)
Lymph node metastasis3533 (94.28)d
Without lymph node metastasis4527 (51.11)
Dukes A to B stage4527 (51.11)e
Dukes C to D stage3533 (94.28)
DISCUSSION

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[1217]. Ezrin protein is an indispensable factor for tumor cell metastasis of osteosarcoma[18], breast cancer[19], nasopharyngeal carcinoma[20], and prostate cancer[21]. In addition, in malignant tumor tissues, there are also changes in subcellular localization of ezrin expression. Moilanen et al[22] 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[23], in the P-gp1-mediated multidrug resistance of cancers, and in cannibalism of metastatic tumors[24]. 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[2526], etc. Ezrin participates in regulating cell-cell and cell-extracellular matrix adhesion, thus influencing tumor cell invasion and other biological behavior[2730]. 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[1]. Pujuguet et al[31] 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[32]. 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[33]. 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.

COMMENTS
Background

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.

Research frontiers

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.

Applications

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.

Terminology

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.

Peer review

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.

Footnotes

Supported by Natural Science Foundation of Shanghai, No. 04ZB14072

References
1.  Swanson KA, Crane DD, Caldwell HD. Chlamydia trachomatis species-specific induction of ezrin tyrosine phosphorylation functions in pathogen entry. Infect Immun. 2007;75:5669-5677.  [PubMed]  [DOI]
2.  Fadiel A, Lee HH, Demir N, Richman S, Iwasaki A, Connell K, Naftolin F. Ezrin is a key element in the human vagina. Maturitas. 2008;60:31-41.  [PubMed]  [DOI]
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]
4.  Fais S. A role for ezrin in a neglected metastatic tumor function. Trends Mol Med. 2004;10:249-250.  [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. Molecular targets for tumour progression in gastrointestinal stromal tumours. Gut. 2004;53:235-240.  [PubMed]  [DOI]
6.  Pang ST, Fang X, Valdman A, Norstedt G, Pousette A, Egevad L, Ekman P. Expression of ezrin in prostatic intraepithelial neoplasia. Urology. 2004;63:609-612.  [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]  [DOI]
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]
11.  Mathew J, Hines JE, Obafunwa JO, Burr AW, Toole K, Burt AD. CD44 is expressed in hepatocellular carcinomas showing vascular invasion. J Pathol. 1996;179:74-79.  [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]  [DOI]
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]
17.  McClatchey AI. Merlin and ERM proteins: unappreciated roles in cancer development? Nat Rev Cancer. 2003;3:877-883.  [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. 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]  [DOI]
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]  [DOI]
22.  Moilanen J, Lassus H, Leminen A, Vaheri A, Bützow R, Carpén O. Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients. Gynecol Oncol. 2003;90:273-281.  [PubMed]  [DOI]
23.  Fais S, De Milito A, Lozupone F. The role of FAS to ezrin association in FAS-mediated apoptosis. Apoptosis. 2005;10:941-947.  [PubMed]  [DOI]
24.  Lugini L, Matarrese P, Tinari A, Lozupone F, Federici C, Iessi E, Gentile M, Luciani F, Parmiani G, Rivoltini L. 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]
27.  Curto M, McClatchey AI. Ezrin...a metastatic detERMinant? Cancer Cell. 2004;5:113-114.  [PubMed]  [DOI]
28.  Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, Mangeat PH, Goldenring JR. Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J. 1997;16:35-43.  [PubMed]  [DOI]
29.  Hunter KW. Ezrin, a key component in tumor metastasis. Trends Mol Med. 2004;10:201-204.  [PubMed]  [DOI]
30.  Yao X, Cheng L, Forte JG. Biochemical characterization of ezrin-actin interaction. J Biol Chem. 1996;271:7224-7229.  [PubMed]  [DOI]
31.  Pujuguet P, Del Maestro L, Gautreau A, Louvard D, Arpin M. Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation. Mol Biol Cell. 2003;14:2181-2191.  [PubMed]  [DOI]
32.  Saras J, Heldin CH. PDZ domains bind carboxy-terminal sequences of target proteins. Trends Biochem Sci. 1996;21:455-458.  [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]