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Clinical Significance of Cellular Distribution of Moesin in Patients with Oral Squamous Cell Carcinoma

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Clinical Significance of Cellular Distribution of Moesin in Patients with Oral Squamous Cell Carcinoma 572 Vol. 10, 572–580, January 15, 2004 Clinical Cancer Research Clinical Significance of Cellular Distribution of Moesin in Patients with Oral Squamous Cell Carcinoma Hiroichi Kobayashi,1 Junji Sagara,2 those with a better prognosis and might improve the defini- Hiroshi Kurita,1 Masayo Morifuji,3 tion of suitable therapy for each. Masamichi Ohishi,3 Kenji Kurashina,1 and Shun’ichiro Taniguchi2 INTRODUCTION Departments of 1Dentistry and Oral Surgery and 2Molecular Oral cancer, 1 of the 10 most common cancers in the world, Oncology and Angiology, Aging and Adaptation, Shinshu University remains a morbid and often fatal disease. Despite marked advances School of Medicine, Matsumoto, Japan and 3First Department of Oral of management and diagnosis of oral squamous cell carcinoma and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University, (OSCC), the overall survival ratio has showed only a modest Fukuoka, Japan increase in recent years. Therefore, the development of molecular markers is needed to improve the diagnosis and assessment of ABSTRACT tumor progression and metastasis in OSCC patients. Moesin is a member of the ERM (ezrin/radixin/moesin) Purpose: Moesin is a linking protein of the submembra- family, which shares ϳ78% amino acid sequence identity with neous cytoskeleton and plays a key role in the control of cell each other. ERM proteins, part of the band 4.1 superfamily, act morphology, adhesion, and motility. The aim of the present as a membrane-cytoskeleton linker in actin-enriched specialized study was to elucidate the clinical significance of expression plasma membrane structures, especially microvilli, ruffling patterns of moesin in patients with oral squamous cell car- membranes, and cleavage furrows and thus play a key role in the cinoma (OSCC). control of cell morphology, adhesion, and motility (1–6). The Experimental Design: Immunohistochemistry for moe- integral membrane proteins such as CD44, CD43, intercellular sin monoclonal antibody was performed on 103 paraffin- adhesion molecules 1 and 2, and actin are identified as ligands embedded specimens from patients with primary OSCC, for ERM proteins (7–9). Merlin, encoded by the neurofibroma- including 30 patients with locoregional lymph node metas- tosis type 2, is classified as a tumor suppressor protein (10). tasis, and in the sections from nude mice transplanted with Because moesin shares high homology with Merlin and colo- two cell lines derived from a single human tongue cancer calizes with it beneath the plasma membrane, it has been spec- (SQUU-A and SQUU-B). ulated that moesin may also be a tumor suppressor (11, 12). Results: Expression patterns of moesin in OSCCs were However, recent studies have indicated that ERM proteins are divided into three groups: membranous pattern; mixed pat- up-regulated in various kinds of tumors (13–17). Whether moe- tern; and cytoplasmic pattern. These expression patterns sin is functional as a tumor suppressor in carcinogenesis and correlated with tumor size, lymph node metastasis, mode of tumor development remains unclear. invasion, differentiation, and lymphocytic infiltration. In Because little is known about the role of moesin in the oral about two-thirds of the patients with metastatic lymph node, normal mucosa and oral lesions, including leukoplakia, verru- homogeneous cytoplasmic expression was detected in the cous carcinoma, and small cell carcinoma, we initiated a series metastatic lymph nodes. In addition, SQUU-B with high of studies aimed at characterizing expression of moesin (18). In metastatic potential showed more reduced levels of mem- this particular study, we report that OSCC cells constitutively brane-bound moesin than SQUU-A with low metastatic po- display several expression patterns of moesin, thereby providing tential. A multivariate analysis demonstrated that expres- a new biomolecular marker for use in prediction of metastasis sion patterns of moesin can be an independent prognostic and poor prognosis. factor. Conclusions: Our results suggest that moesin expres- MATERIALS AND METHODS sion contributed to discriminating between patients with the Patients and Tumor Sample. The study group consisted potentiality for locoregional lymph node metastasis and of 103 patients with OSCC who were diagnosed at the Department of Dentistry and Oral Surgery, Shinshu University School of Med- icine. Tissues of primary (n ϭ 103) and metastatic (n ϭ 30) lesions of OSCCs were collected during biopsy or operation after patients signed the informed consent form approved by the Institution Received 11/1/02; revised 10/14/03; accepted 10/20/03. The costs of publication of this article were defrayed in part by the Review Committee. Permission to perform this study was obtained payment of page charges. This article must therefore be hereby marked from the committee. Thirty-one patients (T1–T2) without lymph advertisement in accordance with 18 U.S.C. Section 1734 solely to node metastasis underwent radiotherapy alone while 72 patients indicate this fact. underwent surgery. The grade of tumor differentiation was deter- Requests for reprints: Hiroichi Kobayashi, Department of Dentistry and Oral Surgery, Shinshu University School of Medicine, Asahi 3-1-1, mined according to the criteria proposed by the WHO (19). Mode Matsumoto 390-8621, Japan. Phone: 81-263-37-2677; Fax: 81-263-37- of invasion was classified according to Jakobsson’s classification 2676; E-mail: [email protected]. (20). Median follow-up time was 32.0 months (range, 2–115 Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 573 Fig. 1 Staining of normal oral epithelium with monoclonal antibody against moesin. A, expression level was gradually reduced from the parabasal layer toward the top layer. B, reactivity for moesin monoclonal antibody was prominent in the cell membrane of basal layer and parabasal layer cells. Bar, 100 ␮minA;10␮minB. months). The study population consisted of 59 men and 44 women Cell Lines and Culture. Human oral cancer cell lines, averaging 65.0 years of age (range, 27–88 years). For controls, SQUU-A and SQUU-B, were established as reported previously normal oral mucosa were obtained from consenting patients during (21). These cell lines were cultured in Eagle’s MEM (Nissui, removal of a lower wisdom tooth. Tokyo, Japan) supplemented with 10% fetal bovine serum (Life Fig. 2 Three staining patterns in oral squamous cell carcinomas with monoclonal antibody against moesin. A, membranous expression pat- tern, predominant expression in the cell membrane. B, mixed expression pattern, reactivity in the cell membrane about equal to that in the cytoplasm. C, cytoplasmic expression pattern, predominant cytoplasmic labeling. Bar,10␮m. Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2004 American Association for Cancer Research. 574 Moesin Expression in Oral Squamous Carcinoma Fig. 3 Immunohistochemical localization of moesin in primary tissue and metastatic lymph nodes of the same oral squamous cell carcinoma patient. A and B, membranous expression of moesin in primary tissue. C and D, cytoplasmic expression in front of an invasive margin of primary tumor cells. E and F, cytoplasmic expression homogeneously observed. Bar, 100 ␮minA, C, and E;10␮minB, D, and F. Technologies, Inc., Grand Island, NY), penicillin (100 IU/ml), zuoka, Japan) and were housed under conventional conditions streptomycin (100 mg/ml), and fungizone (1 mg/ml) at 37°Cin with free access to animal chow and water. Under general ϳ ϫ 5 an atmosphere of 5% CO2. anesthesia with diethylether, 3.5 10 /0.035 ml viable cells Animals and Experimental Treatment. Female were injected in the s.c. tissue of the right side of the tongue. BALB/c mice (6 weeks old) were purchased from SLC (Shi- The mice were sacrificed 5 weeks after the infection, and Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 575 crowaved (500 w, 25 min) for antigen retrieval in 0.01 M citric acid (pH 6.0). After being washed with distilled water and 0.05 M Tris-buffered saline (TBS; pH 7.6), the specimens were treated with mouse anti-moesin antibody diluted by TBS containing 1% BSA at 4°C overnight, washed three times with TBS, and then incubated with goat antimouse immunoglobulin polyclonal antibody diluted by TBS con- taining 1% BSA for 60 min at room temperature. After being washed three times with TBS, the sections were developed in 0.05 M Tris-buffer (pH 7.6) containing 25 mg/125 ml 3,3Ј- diaminobenzidine and 0.0015% hydrogen peroxide for 7 min. The sections were then washed in water, counterstained with Mayer’s hematoxylin, dehydrated, cleaned, and coverslipped. Negative controls were treated by replacing the primary antibody with TBS 1% BSA. Scoring of Results. Sections were examined by two in- dependent researchers (H. Ko., H. Ku.) in an effort to provide a consensus of staining pattern. Moesin or ␤-actin expression of neoplastic cell in primary lesions was classified as follows: membranous pattern—membranous expression of moesin or ␤-actin was more dominant than cytoplasmic expression; mixed pattern—membranous expression of moesin or ␤-actin was ap- Fig. 4 Comparison of percentage of cytoplasmic expression of moesin in primary tissues and metastatic lymph nodes in the same oral squa- proximately equal to cytoplasmic expression; and cytoplasmic mous cell carcinoma patient with cervical lymph node metastasis. In all pattern—membranous expression of moesin or ␤-actin was primary tissues, cytoplasmic expression type was heterogeneously ob- weaker than cytoplasmic expression. We used the expression served (range, 10–92%), whereas in about two-thirds of the metastatic pattern of moesin used as the predominant pattern on the whole lymph nodes, cytoplasmic expression pattern was homogeneously dis- played. histological section of the tumor. tongues were resected. The care and use of these experimental Table 1 Expression pattern of moesin in oral squamous cell animals were in accordance with institutional guidelines.
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