7430

EZR

ezrin;EZR;ezrin

cytovillin 2|epididymis secretory protein Li 105|p81|villin 2 (ezrin)|villin-2

6q25.3

protein-coding

OBJECTIVE: To examine the expression of Ezrin in human salivary gland adenoid cystic carcinoma and investigate the effects of Ezrin gene silence on cell proliferation, apoptosis and invasion of adenoid cystic carcinoma (ACC)-M. METHODS: The expression of Ezrin was detected by immunohistochemistry in normal salivary gland tissue (n=15), pleomorphic adenoma (n=40) and salivary gland adenoid cystic carcinoma (n=43). The Ezrin Stealth RNAi Duplex, containing Stealth RNAi Negative Control Duplex were constructed and transfected into ACC-M cells by Lipofectamine 2000. The expression levels of Ezrin were detected by RT-PCR and immunohistochemistry. The cell cycle and apoptosis rate were analyzed by flow cytometry (FCM). The cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and cell invasion by Transwell test. RESULTS: The positive rate of Ezrin expression in ACC was significantly higher than that in normal salivary gland tissue and pleomorphic adenoma (P<0.05). After transfection of Ezrin Stealth RNAi Duplex, the mRNA and protein expression of Ezrin were down-regulated, the cell proliferation activity was inhibited, the G0-G1 Phase cells were increased, and the apoptosis rate of Ezrin Stealth RNAi Duplex group was higher than that in control groups and cell invasion ability was decreased. CONCLUSIONS: Over expression of Ezrin in human salivary gland adenoid cystic carcinoma may promote genesis, development and metastasis of tumors. Ezrin Stealth RNAi Duplex could efficiently down-regulate the expression of Ezrin gene, and partly inhibited proliferation of ACC-M cells, induce apoptosis and decrease invasion ability of these cells in vitro.

OBJECTIVE: The aim of this study was to investigate the relationship between membrane cytoskeleton linker protein Ezrin and CD44v6, iNOS, Ki-67, and clinicopathologic characteristics, and the prognostic significance of Ezrin expression in salivary gland adenoid cystic carcinoma (SACC). STUDY DESIGN: Immunohistochemistry and reverse-transcription polymerase chain reaction were used to quantify the expression of Ezrin, CD44v6, inducible nitric oxide synthase (iNOS), and Ki-67 in 75 primary SACCs, 25 tumor-free salivary tissues, and 2 SACC cell lines (ACC-M and ACC-2). Survival analysis was performed to find the prognostic significance of Ezrin expression. RESULTS: expressions of Ezrin, CD44v6, iNOS, and Ki-67 in SACC tissues, especially with distant metastasis, were significantly higher than in tumor-free tissues. Ezrin mRNA and protein levels in ACC-M cells were significantly higher than in ACC-2 cells. Ezrin, CD44v6, iNOS, and Ki-67 expressions were significantly higher in solid pattern than in cribriform and tubular patterns. Ezrin and its partners, CD44v6, iNOS, and Ki-67, were significantly related to tumor size, clinical stage, perineural and vascular invasion, and recurrence. Furthermore, Ezrin had an independent prognostic effect on overall survival. CONCLUSIONS: The increased expression of Ezrin and its partners, CD44v6, iNOS, and Ki-67, in SACC correlated with histologic patterns, may play a role in distant metastasis, and might indicate poor clinical outcome.

Ezrin primarily acts as a linker between the plasma membrane and the cytoskeleton and is a key component in tumor metastasis. In the present study, RNA interference (RNAi) using ezrin small hairpin RNAs (ezrin shRNAs) was used to define the roles of ezrin in the regulation of malignant behaviors of human breast cancer. The highly metastatic human breast cancer cell MDA-MB-231, in which ezrin mRNA and protein levels are the highest, was selected as a cell model in vitro. In addition, we also found that ezrin expression was up-regulated and its immuno-staining trans-located from cell membrane to cytoplasm, whereas E-cadherin expression decreased and showed the same cell distribution as ezrin in lymphatic metastases of human breast carcinomas. After repression of ezrin by more than 85% of G3PDH and 75% of beta-actin in mRNA and protein levels was maintained in the stable expressing ezrin shRNAs MDA-MB-231 cell clones, the abilities of cell motility and invasiveness were obviously inhibited with a 4-fold and 2-fold, respectively, and the altered cell polarity was observed. Western blot analyses further revealed that the silencing of ezrin induced an increased E-cadherin expression and a decreased phosphorylation of beta-catenin by inhibiting phosphorylation levels of c-src. These data indicate that ezrin overexpression positively correlated with metastatic potentials of human breast cancer cells, especially lymphatic system metastasis. Decreased ezrin expression by shRNA reversed metastatic behaviors of human breast cancer cells by inducing c-src-mediated E-cadherin expression, suggesting that ezrin may have potential values in assessing lymphatic metastasis of human breast cancers.

BACKGROUND AND OBJECTIVE: Both RhoA and Ezrin are confirmed to play an important role in the development and metastasis of tumors. However, the mechanism remains unclear. This study rudimentally investigates the regulatory effect of RhoA on the expression of Ezrin. METHODS: After MDA-MB-231 cells were treated with epidermal growth factor (EGF) or following pretreatment of fasudil (the special inhibitor of RhoA), the expression of RhoA, p-RhoA and Ezrin in MDA-MB-231 cells was detected by western blot. RESULTS: Stimulation of EGF triggered RhoA phosphorylation in MDA-MB-231 cells which reached the maximum at 30 min; RhoA expression did not change; Ezrin expression was enhanced and reached the maximum at 24 h. However, pretreatment of fasudil before EGF stimulation decreased RhoA phosphorylation and Ezrin expression in MDA-MB-231 cells by 72.73% and 51.28%, respectively. CONCLUSION: RhoA may regulate the invasion and metastasis of breast cancer cells as an upstream signaling of Ezrin.

This study was undertaken to observe the effects and the possible mechanism of membrane-cytoskeleton linker ezrin on the chemokine CC chemokine ligand 5 (CCL5)-induced invasive ability in human breast carcinoma MCF-7 cells. RNA interference (RNAi) using ezrin small hairpin RNAs (ezrin shRNA) was used to analyze the role of ezrin in the regulation of this CCL5-induced malignant behavior of MCF-7 cells. The effects of recombinant human CCL5 (rhCCL5) on the cell's invasive ability were detected by transwell assay. Western blotting was performed to examine the expression of the total and the phosphorylated ezrin at protein level. The CCL5-induced changes in the organization of the actin cytoskeleton in the transfected cells were determined using confocal microscopy. Flow cytometry was used to detect the cell cycle. MTT method was used to detect the proliferation of the cells. We found that the MCF-7 cells responded chemotactically to CCL5 in a dose- and time-dependent manner. After RNAi treatment, the proliferation was inhibited and the cell proportion in G2-M phase decreased. The CCL5-induced cell motility and invasiveness were obviously inhibited by the silencing of ezrin. In addition, the CCL5 induced a significant up-regulation in the total and the phosphorylated ezrin expression in MCF-7 cells, whereas in the presence of ezrin silencing, the CCL5 induced only a slight increase in the total and the phosphorylated ezrin expression. CCL5 was shown to induce changes in the organization of the actin cytoskeleton and the level of F-actin in MCF-7 cells, and the silencing of ezrin could inhibit these changes. Collectively, our data further show that CCL5 induced invasiveness in MCF-7 cells. These data indicate a potential role for ezrin in the processes of the CCL5-induced breast cancer cell migration, invasiveness and metastasis. It is suggested that ezrin may act as downstream effector of CCL5 and a new anti-invasive therapeutic target for human breast cancer.

AIMS AND BACKGROUND: Overexpression of ezrin contributes to the progression and invasiveness of several human cancers; however, its role in breast cancer metastasis has not been investigated in detail. METHODS: Ezrin expression in tissue samples from patients with invasive ductal carcinoma of the breast was detected by immunohistochemistry. Ezrin expression in a breast cancer cell line was evaluated using Western blot and RT-PCR. RESULTS: Elevated expression of ezrin was associated with lymph node metastasis and poor prognosis in patients with invasive ductal carcinoma. Ezrin expression was related to the invasiveness of breast cancer cells in vitro. Low-dose epirubicin inhibited the migration of breast cancer cells in a concentration-dependent manner without promoting cytotoxicity in vitro and decreased the expression of ezrin in a concentration-dependent manner. CONCLUSIONS: Low-dose epirubicin may be antimetastatic without promoting cytotoxic effects and could serve as a target for the development of therapeutics for breast carcinoma.

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.

Hyperactivation of beta-catenin-T-cell-factor (TCF)-regulated gene transcription is a hallmark of colorectal cancer (CRC). The cell-neural adhesion molecule L1CAM (hereafter referred to as L1) is a target of beta-catenin-TCF, exclusively expressed at the CRC invasive front in humans. L1 overexpression in CRC cells increases cell growth and motility, and promotes liver metastasis. Genes induced by L1 are also expressed in human CRC tissue but the mechanisms by which L1 confers metastasis are still unknown. We found that signaling by the nuclear factor kappaB (NF-kappaB) is essential, because inhibition of signaling by the inhibitor of kappaB super repressor (IkappaB-SR) blocked L1-mediated metastasis. Overexpression of the NF-kappaB p65 subunit was sufficient to increase CRC cell proliferation, motility and metastasis. Binding of the L1 cytodomain to ezrin - a cytoskeleton-crosslinking protein - is necessary for metastasis because when binding to L1 was interrupted or ezrin gene expression was suppressed with specific shRNA, metastasis did not occur. L1 and ezrin bound to and mediated the phosphorylation of IkappaB. We also observed a complex containing IkappaB, L1 and ezrin in the juxtamembrane region of CRC cells. Furthermore, we found that L1, ezrin and phosphorylated p65 are co-expressed at the invasive front in human CRC tissue, indicating that L1-mediated activation of NF-kappaB signaling involving ezrin is a major route of CRC progression.

AIM: To investigate the early metastasis-associated proteins in sentinel lymph node micrometastasis (SLNMM) of colorectal cancer (CRC) through comparative proteome. METHODS: Hydrophobic protein samples were extracted from individual-matched normal lymph nodes (NLN) and SLNMM of CRC. Differentially expressed protein spots were detected by two-dimensional electrophoresis and image analysis, and subsequently identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry-mass spectrometry and Western blotting, respectively. RESULTS: Forty proteins were differentially expressed in NLN and SLNMM, and 4 metastasis-concerned proteins highly expressed in SLNMM were identified to be hnRNP A1, Ezrin, tubulin beta-2C and Annexin A1. Further immunohistochemistry staining of these four proteins showed their clinicopathological characteristics in lymph node metastasis of CRC. CONCLUSION: Variations of hydrophobic protein expression in NLN and SLNMM of CRC and increased expression of hnRNP A1, Ezrin, tubulin beta-2C and Annexin A1 in SLNMM suggest a significantly elevated early CRC metastasis.

OBJECTIVE: To evaluate the expression of ezrin and CD44-v6 in esophageal squamous cell carcinoma, and to evaluate its relationship with lymph node metastasis (LNM) and histological grading. METHODS: The expression of ezrin and CD44-v6 in 71 patients with esophageal squamous cell carcinoma was studied using immunohistochemical (SP) method. The correlation of their expression with relevant clinical data was statistically analyzed. RESULTS: In normal esophageal squamous epithelia, the expression of ezrin was found in 33 cases among 71 cases and the expression of CD44-v6 in 18 cases among 71 cases. In esophageal squamous cell carcinoma, the expression of ezrin was found in 64 cases among 71 cases and CD44-v6 in 58 cases among 71 cases. The expression of ezrin was closely related to LNM. The positive rate of ezrin expression in LNM cases was significantly higher than that in cases without LNM. The expression of CD44-v6 had a close relation to tumor differentiation and LNM. The positive rate of CD44-v6 expression in LNM cases was significantly higher than that in patients without LNM. The expression of ezrin in CD44-v6 positive cases was significantly higher than that of CD44-v6 negative cases. The LNM rate was 60.0% in 48 patients with positive expression of both ezrin and CD44-v6, while none of lymph node metastasis was found in the 6 patients with both negative. CONCLUSION: The test of CD44-v6 and ezrin expression may have significant prognostic value for assessing the degree of malignancy and potential LNM probability of ESCC. Ezrin may become a new target in evaluation of tumor prognosis.

Gastroesophageal reflux has recently been implicated as a causative factor in upper aerodigestive tract carcinogenesis. Esophageal squamous cell carcinomas (ESCCs) have developed in duodenal-content reflux animals without any known carcinogen present. We established a cell line, designated ESCC-DR, from a thoracic metastatic tumor in a reflux animal. To gain insight into the genomic alterations associated with duodenal content reflux-induced carcinogenesis, we first performed comparative genomic hybridization using an Agilent rat 244K array in ESCC-DR and identified many chromosomal gains and losses. Of the many genes identified, we detected an interesting ezrin amplicon that has been recently reported in human ESCC. Ezrin, which cross-links the cytoskeleton and plasma membrane, is involved in the growth and metastatic potential of cancer cells. Overexpression of ezrin protein in ESCC-DR was confirmed by Western blotting. We also compared ezrin protein expression levels and patterns in hyperplastic, dysplastic, ESCC, and metastatic sites developed in two distinct reflux models using immunohistochemistry. Immunohistochemical staining of ezrin revealed overexpression in the nucleus, and the cytoplasm as well as plasma membrane of ESCC cells. Phosphorylated ERM (ezrin, radixin, moesin) was expressed at the leading edge, or invasive front, of larger metastatic sites. Taken together, duodenal reflux has a great potential for initiating malignancy, and thus likely plays a role in development of ESCC. Ezrin probably influences the growth and invasiveness of ESCC cells, and phosphorylation is only required in metastatic behavior of tumor cells at the leading edge and invasive front.

BACKGROUND AND OBJECTIVE: It has been proven that Ezrin protein may interact with E-cadherin protein and take part in metastasis of tumor cells. This study was to investigate the expressions of Ezrin and E-cadherin in esophageal squamous cell carcinoma (ESCC) and their relationship with the clinicopathologic factors, and analyze their diagnostic values for ESCC. METHODS: The expression of Ezrin and E-cadherin in 72 specimen of ESCC and the paracancer normal squamous epithelium was detected using tissue array with SP immunohistochemistry. Their correlations to the clinicopathologic factors were analyzed statistically. RESULTS: The positive rate of Ezrin was significantly higher in ESCC than in para-cancer normal squamous epithelium (90.7% vs. 46.0%, P < 0.001); the positive rate of E-cadherin was significantly lower in ESCC than in para-cancer normal squamous epithelium (27.6% vs. 97.4%, P < 0.001). Ezrin expression was related to the invasiveness and lymph node metastasis of ESCC (P < 0.05); E-cadherin expression was related to the differentiation and lymph node metastasis of ESCC (P < 0.05). The high expression of Ezrin was related to the low expression of E-cadherin (P < 0.05). CONCLUSION: The activation of Ezrin and the absence of E-cadherin contribute to the tumorigenesis and metastasis of ESCC.

Three closely related proteins, ezrin, radixin, and moesin (ERM), which primarily act as a linker between the plasma membrane and the cytoskeleton, are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. Although, ezrin is now recognized as a key component in tumor metastasis, the functional role of the radixin and moesin in tumor metastasis has not been established. In the present study, we chose highly metastatic human gastric carcinoma SGC-7901 cells, which express all of the ERM proteins as a model to examine the functional roles of these proteins in tumor metastasis. Ezrin, radixin or moesin stable knockdown SGC-7901 cell lines were established using siRNA methodology. In vitro cell migration and invasion studies showed that either ezrin, radixin or moesin specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. Western blotting and immunofluorescence analysis showed that the expression of E-cadherin was also significantly increased when any member of ERM proteins was downregulated. Our results indicated that these three ERM proteins play similar roles in the SGC-7901 cell metastatic potential and their roles of upregulating the expression of E-cadherin may be important in tumor progression.

OBJECTIVE: To investigate the relationship of ezrin protein expression to the carcinogenesis and prognosis of infiltrating breast ductal carcinoma. METHODS: S-P immunohistochemical staining was used to detect the ezrin protein expression in 88 patients with infiltrating ductal carcinoma, and in 54 patients with intraductal hyperplastic lesions of the breast. The clinicopathological data and follow-up information of these patients were all obtained. The relationship of ezrin protein expression to the clinicopathological parameters and the prognostic significance in the infiltrating breast ductal carcinoma was analyzed using Chi-square test (chi2), Kaplan-Meier and Cox models. RESULTS: The immunohistochemical staining results showed that the strong positive expression rate of ezrin protein in the normal ductal epithelium, simple ductal hyperplasia, atypical hyperplasia and infiltrating ductal carcinoma of the breast was 9.1%, 16.7%, 43.3% and 64.8%, respectively, which was significantly higher in atypical hyperplasia and infiltrating ductal carcinoma than that in the normal ductal epithelium and simple ductal hyperplasia (P < 0.05). The strong ezrin protein expression in the infiltrating ductal carcinoma was positively correlated with axillary lymph node metastasis, histological differentiation grade, TNM stage and CD44v6 expression, but negatively correlated with the expression of E-cadherin (P < 0.05). It was also found that the survival of the patient with strong positive expression of ezrin protein was significantly shorter than that of the control (P < 0.05). CONCLUSION: Ezrin protein may play an important role in the carcinogenesis of infiltrating breast ductal carcinoma. The strong expression of ezrin protein may be used as a biomarker for predicting poor prognosis in the patients with infiltrating breast ductal carcinoma.

CONCLUSION: The overexpression of HMGA1 or Ezrin may contribute to the carcinogenesis, development, and metastasis of laryngeal squamous cell carcinoma (LSCC). OBJECTIVE: To investigate the expression of HMGA1 and Ezrin in LSCC and analyze their clinical significance. METHODS: The expression of HMGA1 and Ezrin was analyzed by immunohistochemistry (IHC) in 50 cases of LSCC. Thirty cases of laryngeal polyp and 30 cases of atypical hyperplasia of larynx were studied as controls. The expression of HMGA1 and Ezrin was analyzed by real-time PCR and by Western blot in 30 cases of LSCC; samples from adjacent normal epithelial tissues in 30 cases were studied as controls. RESULTS: (1) IHC revealed that the positive rate of HMGA1 protein was 68.0% (34/50), 53.3% (16/30), and 13. 3% (4/30) in LSCC, atypical hyperplasia of larynx, and laryngeal polyp (p < 0.05), and the positive rate of Ezrin protein was 64.0% (32/50), 50.0% (15/30), and 23.3% (7/30) (p < 0.01), respectively. (2) Real-time PCR demonstrated that the mean relative mRNA expression levels of HMGA1 in LSCC and in normal tissues were 2.41 +/- 0.40 and 1.05 +/- 0.18, respectively (p < 0.01). The mRNA levels of Ezrin in LSCC and in normal tissues were 1.79 +/- 0.27 and 1.04 +/- 0.22, respectively (p < 0.05). (3) Western blotting revealed that the mean relative protein expression levels of HMGA1 in LSCC and in normal tissues were 1.73 +/- 0.60 and 0.35 +/- 0.17, respectively (p < 0.01). The protein levels of Ezrin in LSCC and in normal tissues were 1.82 +/- 0.77 and 0.42 +/- 0.20, respectively (p < 0.01).

OBJECTIVE: To investigate the different expressions of cytoskeletal organizer ezrin and cytoskeleton protein beta- and gamma-actin in hepatocellular carcinoma (HCC) cell lines with different metastatic potentials and to explore the role of ezrin in cell growth and metastasis in HCC cell lines SF7721 and MHCC97-H. METHODS: Immunofluorescence, RT-PCR and Western blot were used to detect the gene and protein expressions of ezrin and actin in hepatocellular carcinoma cell lines with different metastatic potentials. RNA interference (RNAi) was applied to down-regulate the ezrin expression in SF7721 and MHCC97-H. Changes of the cell growth and metastasis potentials after the RNAi treatment were studied. MTT assay was used to detect cell proliferation changes and Transwell assay was applied to observe the changes of cell motility and invasiveness. RESULTS: Both ezrin and cytoskeleton protein were demonstrated in the cytoplasma of the cells at the same time. The expression of them in cell lines with high metastatic potential, such as SF7721, MHCC-1 and MHCC97-H was obviously higher than in those with low metastatic potentials, such as SMMC-7721, Hep3B and HepG2 (chi2= 13.277, P = 0.010; chi2= 21.815). The mRNA and ezrin and cytoskeleton protein gamma-actin were over-expressed in HCC cell lines with high metastatic potentials. The expressions of beta-actin of cell lines with different metastatic potentials showed no differences. Ezrin protein was successfully down-regulated and the proliferation and the invasiveness of the cells decreased with low ezrin protein level in SF7721 and MHCC97-H. CONCLUSION: Over-expression of ezrin and cytoskeleton protein gamma-actin are associated with the process of metastasis of hepatocellular carcinoma cells. The growth and invasiveness of SF7721 and MHCC97-H cells can be inhibited by down-regulating ezrin expression.

OBJECTIVE: expression of microRNA-22 (miR-22) and ezrin protein (a membrane-cytoskeleton linking protein) in hepatocellular carcinoma (HCC) was investigated. METHODS: Specimens of HCC and paracancerous tissue (control; ~5 cm away from tumour tissue) were collected from 192 patients. miR-22 expression was detected by real-time polymerase chain reaction; ezrin protein expression in tumour tissue was assessed immunohistochemically. Associations between miR-22 expression and clinicopathological features of HCC and ezrin expression were analysed. RESULTS: miR-22 expression was lower in HCC tissue than in paracancerous tissue samples (median relative expression 0.676 versus 1.000 for control tissue). expression of miR-22 was significantly associated with histological differentiation (relative expression 0.431 for lower grades of differentiation versus 0.918 for higher grades), and was associated with lymphatic metastasis (relative expression 0.518 if metastasis was present, 0.919 if absent). Survival time was shorter in patients with low miR-22 expression than in those with high expression (31.0 +/- 2.6 versus 52.2 +/- 5.1 months). There was a significant negative correlation between the expression of miR-22 and that of ezrin. CONCLUSIONS: miR-22 is downregulated in HCC and its expression is associated with the differentiation, metastasis and prognosis of the carcinoma. Ezrin is a potential regulatory protein of miR-22.

Lung cancer often metastasizes to bone in patients with advanced disease. Identification of the factors involved in the interactions between lung cancer cells and bone will improve the prevention and treatment of bone metastases. We identified changes in metastasis-related gene expression of human HARA lung squamous carcinoma cells co-cultured with neonatal mouse calvariae using a pathway-specific microarray analysis. Nine genes were up-regulated and two genes down-regulated in HARA cells co-cultured with mouse calvariae. Five of the nine up-regulated genes, including caveolin 1, CD44, EphB2, ezrin, and Parathyroid hormone-related protein (PTHrP), and one down-regulated gene, SLPI, were further confirmed by Reverse transcription-polymerase chain reaction (RT-PCR). A mouse model was subsequently used to study the role of PTHrP and ezrin in bone metastasis in vivo. PTHrP (all three isoforms) and ezrin were up-regulated in HARA cells at sites of bone metastasis as detected by RT-PCR and immunohistochemistry. The PTHrP 141 mRNA isoform was increased by the greatest extent (13.9-fold) in bone metastases compared to PTHrP 139 and PTHrP 173 mRNA. We then generated a HARA cell line in which PTHrP expression was inducibly silenced by RNA interference. Silencing of PTHrP expression caused significant reduction of submembranous F-actin and decreased HARA cell invasion. Ezrin up-regulation was confirmed by Western blots on HARA cells co-cultured with adult mouse long bones. Further, Transforming growth factor beta (TGF-beta) was identified as one of the factors in the bone microenvironment that was responsible for the up-regulation of ezrin. The identification of PTHrP and ezrin as important regulators of lung cancer bone metastasis offers new mechanistic insights into the metastasis of lung cancer and provides potential targets for the prevention and treatment of lung cancer metastasis.

Lung cancer is the leading cause of cancer death. However, the mechanism of lung cancer relapse and metastasis has been poorly elucidated. Recent researches have addressed the role of MicroRNAs (miRNAs) in mediating tumor metastasis. In the present study, we identified microRNA-183 (miR-183) as a potential metastasis-inhibitor. expression level of miR-183 was reversely correlated with the metastatic potential of lung cancer cells. Furthermore, over-expression of miR-183 inhibited migration and invasion of lung cancer cells. Mechanistically, we identified VIL2-coding-protein Ezrin as a bona fide target of miR-183. We also found that miR-183 could regulate the expression of other genes involved in migration and invasion. Taken together, our findings demonstrated a new role and regulatory mechanism of miR-183 in controlling cancer metastasis.

Ezrin, one of the ezrin-radixin-moesin proteins, is involved in the formation of cell membrane processes such as lamellipodia and filopodia and acts as a membrane-cytoskeleton linker. Its aberrant expression correlates with development and progression of several human cancers. However, the expression of ezrin and its role in lung cancer are currently unknown. In this study, we performed ezrin small interfering RNA transfection in two lung cancer cell lines and examined the effects on malignant phenotypes in cancer cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and chamber transwell assays. Ezrin knockdown significantly reduced the proliferation, migration, and invasion of lung cancer cells in vitro. To address the possible mechanisms, we evaluated the expression of adhesion molecules E-cadherin and beta-catenin by Western blot and reverse transcriptase-polymerase chain reaction analyses. The results demonstrated that downregulation of ezrin reduced beta-catenin and increased E-cadherin at the protein level but had no effects on their mRNA levels, suggesting posttranscriptional regulation of these two adhesion molecules. Immunofluorescence assays revealed that ezrin knockdown restored membranous expression of E-cadherin and decreased cytoplasmic beta-catenin in lung cancer cells. In addition, ezrin expression was immunohistochemically evaluated on 135 normal and 183 lung cancer tissues. The expression of ezrin was significantly higher in cancer samples than paired autologous normal lung tissues. In normal bronchial epithelium, ezrin was mainly localized on the apical membrane, while in lung cancers and metastatic foci, ezrin was primarily distributed in cytoplasm. Among lung cancer tissues, expression of ezrin was higher in the invasive front of primary lesions and the highest in lymphatic metastasis. Statistical analysis demonstrated that ezrin expression correlated significantly with lymphatic metastasis and advanced TNM stage. Our data suggest that ezrin may play a crucial role in governing the biological behavior of lung cancer.

Ezrin, primarily acts as a linker between the plasma membrane and the cytoskeleton, is involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion, motility, and modulation of signaling pathways. Although ezrin is now recognized as a key component in tumor metastasis, its roles and the underlying mechanisms remain unclear. In the present study, we chose highly metastatic human lung carcinoma 95D cells, which highly express the ezrin proteins, as a model to examine the functional roles of ezrin in tumor suppression. An ezrin-silenced 95D cell line was established using lentivirus-mediated short hairpin RNA method. CCK-8 assay and soft agar assay analysis showed that downregulation of ezrin significantly suppressed the tumorigenicity and proliferation of 95D cells in vitro. cell migration and invasion studies showed that ezrin-specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. In parallel, it also induced rearrangements of the actin cytoskeleton. Flow cytometry assay showed that changes in the ezrin protein level significantly affected the cell cycle distribution and eventual apoptosis. Furthermore, further studies showed that ezrin regulated the expression level of E-cadherin and CD44, which are key molecules involved in cell growth, migration, and invasion. Meanwhile, the suppression of ezrin expression also sensitized cells to antitumor drugs. Altogether, our results demonstrated that ezrin played an important role in the tumorigenicity and metastasis of lung cancer cells, which will benefit the development of therapeutic strategy for lung cancer.

CD44 is a cell adhesion molecule that was traditionally known as 'homing receptor'. This molecule is known to interact with the ezrin family (ERM family) members and form a complex that plays diverse roles within both normal and abnormal cells, particularly cancer cells. CD44 and ezrin and their respective complex have properties suggesting that they may be important in the process of tumour-endothelium interactions, cell migrations, cell adhesion, tumour progression and metastasis. This article reviews the role of CD44, ezrin family and the CD44/ezrin complex in cancer cells and their clinical impact in patients with cancer.

We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis.

Desmoglein 3 (Dsg3), the pemphigus vulgaris antigen, has recently been shown to be upregulated in squamous cell carcinoma (SCC) and has been identified as a good tumor-specific marker for clinical staging of cervical sentinel lymph nodes in head and neck SCC. However, little is known about its biological function in cancer. The actin-binding protein Ezrin and the activator protein 1 (AP-1) transcription factor are implicated in cancer progression and metastasis. Here, we report that Dsg3 regulates the activity of c-Jun/AP-1 as well as protein kinase C (PKC)-mediated phosphorylation of Ezrin-Thr567, which contributes to the accelerated motility of cancer cells. Ectopic expression of Dsg3 in cancer cell lines caused enhanced phosphorylation at Ezrin-Thr567 with concomitant augmented membrane protrusions, cell spreading and invasive phenotype. We showed that Dsg3 formed a complex with Ezrin at the plasma membrane that was required for its proper function of interacting with F-actin and CD44 as Dsg3 knockdown impaired these associations. The increased Ezrin phosphorylation in Dsg3-overexpressing cells could be abrogated substantially by various pharmacological inhibitors for Ser/Thr kinases, including PKC and Rho kinase that are known to activate Ezrin. Furthermore, a marked increase in c-Jun S63 phosphorylation, among others, was found in Dsg3-overexpressing cells and the activation of c-Jun/AP-1 was further supported by a luciferase reporter assay. Taken together, our study identifies a novel Dsg3-mediated c-Jun/AP-1 regulatory mechanism and PKC-dependent Ezrin phosphorylation that could be responsible for Dsg3-associated cancer metastasis.

A significant proportion of patients with nasopharyngeal carcinoma (NPC) will develop regional relapse or distant metastasis after treatment. So, to find valuable prognostic factors becomes very important. To elucidate the expression of ezrin in NPC and its correlation with patients' clinicopathologic characteristics, time to progression, and overall survival, immunohistochemistry stains for ezrin were applied to 200 tissue specimens of NPC. Ezrin expression had no significant correlation with sex, age, primary tumor stage (T), Epstein-Barr virus (EBV) VCA-IgA antibody, EBV EA-IgA antibody, and EBV DNA copy except primary nodal status (N; p= .032). Increased ezrin expression was significantly related to higher lymph nodal metastatic rate (p< .004). Among ezrin (negative/weak), ezrin (moderate), and ezrin (intense) groups, there was a significant difference for median time to progression (not reached vs. 63.2 months vs. 46 months, P< .001) and median overall survival (100.7 months vs. 80.8 months vs. 70.6 months, P< .001). Ezrin expression status was an independent prognostic factor in multivariate (Cox) analysis. Ezrin, which was significantly correlated to prognosis of disease, may be involved in the tumorigenesis, progression, and invasion of NPC. Thus, our data offer the possibility of future therapeutic targets.

N,N'-Dinitrosopiperazine (DNP) is a carcinogen for nasopharyngeal carcinoma (NPC), which shows organ specificity to nasopharyngeal epithelium. Herein, we demonstrate that DNP induces fiber formation of NPC cells (6-10B) and also increases invasion and motility of 6-10B cells. DNP-mediated NPC metastasis also was confirmed in nude mice. Importantly, DNP induced the expression of phosphorylated ezrin (phos-ezrin) at threonine 567 (Thr-567) dose- and time-dependently but had no effect on the total ezrin expression at these concentrations. Furthermore, DNP-induced phos-ezrin expression was dependent on increased Rho kinase and protein kinase C (PKC) activity. DNP may activate Rho kinase through binding to its pleckstrin homology and may activate PKC through promoting its translocation to the plasma membrane in vivo. DNP-induced phos-ezrin was associated with induction of fiber growth in 6-10B cells. However, DNP could not induce motility and invasion of NPC cells containing ezrin mutated at Thr-567. Similarly, DNP could not induce motility and invasion of the cells containing siRNAs against Rho or PKC. These results indicate that DNP induces ezrin phosphorylation at Thr-567, increases motility and invasion of cells, and promotes tumor metastasis. DNP may be involved in NPC metastasis through regulation of ezrin phosphorylation at Thr-567.

PURPOSE: The primary objective of this study was to examine ezrin in the CD44(+) subpopulation of SCC-9 cells in a malignant oral cancer cell line. MATERIALS AND METHODS: The CD44(+) subpopulation of SCC-9 cells was sorted using CD44 fluorescence-activated cell sorting flow cytometry. expressions of the ABCG2, CD133, Oct-4, BMI-1, an ezrin genes and proteins were detected by immunofluorescence and real-time polymerase chain reaction, and their differential expressions in the CD44(+) and CD44(-) cell populations and embryonic stem cells were compared. The location of the ezrin protein expressed in SCC-9 cells was determined using laser scanning confocal microscopy. RESULTS: The expression of ezrin in the CD44(+) subpopulation was 38 times higher than in the CD44(-) subpopulation based on real-time polymerase chain reaction, and levels of ABCG2, CD133, Oct-4, and BMI-1 in the CD44(+) subpopulation were higher than those in the CD44(-) subpopulation (P < .05). CONCLUSION: The overexpression of ezrin in the CD44(+) subpopulation of SCC-9 cells might affect the functions of CD44(+) tumor cells, which contribute to the metastatic behavior of malignant oral squamous cell carcinoma.

Ezrin is a cytoskeleton linker protein that is actively involved in the regulation of growth and metastatic capacity of cancer cells. Recently, it has been demonstrated that a significant correlation exists between high ezrin expression levels and the poor outcome of pediatric osteosarcoma patients. The expression of ezrin was compared in conventional high-grade and central low-grade osteosarcoma lesions to investigate the role of ezrin overexpression in the metastasis of osteosarcoma. We compared the expression levels of the ezrin protein in 32 cases of high-grade osteosarcomas and 21 cases of low-grade osteosarcomas using immunohistochemistry. Ezrin protein expression levels were examined in three different human osteosarcoma cell lines by Western blotting. In addition, the mRNA expression levels of ezrin in these osteosarcoma cell lines and control fibroblasts were evaluated by real-time quantitative PCR. Ezrin immunoreactivity was present in 43.7% of high-grade osteosarcoma specimens. All low-grade osteosarcomas were negative for ezrin. The expression of ezrin was detected by Western blotting in all three osteosarcoma cell lines. The tested osteosarcoma cell lines showed marked amplification of ezrin mRNA compared to control cells. Taken together, ezrin appears to play a role in the progression of tumors, such as the metastasis of osteosarcoma. However, further data are needed before ezrin can be considered in clinical decision-making about osteosarcoma patients.

BACKGROUND: Ezrin is a membrane-cytoskeleton linker protein involved in regulating growth and metastatic behaviour of cancer cells. The study evaluated ezrin expression and its prognostic value in patients with non-metastatic osteosarcoma. PROCEDURES: Ezrin expression and pattern of staining (cytoplasmic or membraneous and cytoplasmic) were assessed using immunohistochemistry on slides from tumour biopsy. We studied 95 patients (median 16 years, range: 4-39 years) with primary non-metastatic osteosarcoma of the extremity treated by neoadjuvant chemotherapy based on methotrexate, cisplatin, doxorubicin and ifosfamide. RESULTS: Seventy-six patients (80%) showed ezrin immunoreactivity: in cytoplasm (37, 47%) and in cytoplasm and membrane (42, 53%) of tumour cells. Immunohistochemical staining score was: 1+ (16, 24%), 2+ (10, 13%), 3+ (17, 21.5%) and 4+ (36, 45.5%). Ezrin and score expression were not related to gender, site, alkaline phosphatase (AP), LDH serum levels, chemotherapy-induced tumour necrosis or patient outcome. A significant association was seen between expression pattern and prognosis. The 3-year probability of disease-free survival was 80% for patients with only cytoplasmic immunostaining and 54% for patients with cytoplasmic and membranous immunostaining (P < 0.02). CONCLUSION: Ezrin immunoreactivity can be detected in the majority of patients with non-metastatic osteosarcoma of the extremity. The pattern of ezrin staining can identify patients with different risks of relapse. In patients who only have ezrin cytoplasmic expression, a probability of EFS >80% at 5 years can be expected. These results suggest further investigations to define the relation between expression pattern, ezrin functional status and outcome in patients with non-metastatic osteosarcoma.

Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCalpha, PKCiota and PKCgamma. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

PURPOSE: To determine the prognostic value of the expression of Ezrin, CD44 and Six1 genes in osteosarcoma tissues of Chinese patients. METHODS: Fluorescent quantitative real-time PCR was applied to study the mRNA levels of Ezrin, CD44 and Six1 genes in 32 osteosarcoma patient samples and 10 adjacent normal tissues and MG63 osteosarcoma cell lines. The analysis of relationships between pulmonary metastasis and overall survival time were carried out based on the clinical data. RESULTS: mRNA levels of Ezrin and Six1 genes in osteosarcoma tissues were higher than those in adjacent normal tissues (P=0.015, 0.025). The mRNA levels of Ezrin, CD44 and Six1 genes were closely correlated with Enneking GTM clinical staging, while no correlations were demonstrated between the mRNA level of these genes with sex, age, location or pathological types. In addition, we demonstrated that the high mRNA level of Ezrin gene was related to shorter lung metastasis-free and overall survival time of the Chinese patients with osteosarcoma (P < 0.001). CONCLUSION: Our data suggest that Ezrin, but not CD44 and Six1, could be a prognostic factor and a predictor of potential lung metastasis in osteosarcoma. Further large sample studies need to be done to confirm the potential value of Ezrin as a new therapeutic target.

AIMS AND BACKGROUND: Ezrin is a membrane-cytoskeleton linker protein involved in regulation of the growth and metastatic behavior of cancer cells. metastatic tumor antigen (MTA) is a potential metastasis-associated protein. The objective of this study was to evaluate the expression of ezrin and MTA and their correlation with clinicopathological features in osteosarcomas of the jaw. METHODS: We analyzed ezrin and MTA protein levels by immunohistochemistry in 31 osteosarcomas of the jaw. RESULTS: The mean age at diagnosis was 39 years and half of the patients were male. The mandible (n = 19) was more frequently involved than the maxilla (n = 12). The predominant histological type was chondroblastic (58.1%) and 24 patients (77.4%) were classified as having a high grade of malignancy. Immunoreactivity for ezrin was identified in 6 of 31 cases (19.4%), while 77.4% displayed expression of MTA. All ezrin-positive patients had high-grade tumors. The high-grade tumors (n = 24) had a higher rate of MTA expression (42.9% vs 87.5%). expression of ezrin and MTA was not significantly different according to age, sex, tumor site, histological type, and tumor ploidy. Follow-up information was available for 13 patients, with a mean follow-up time of 26.7 months (range, 6-48 months). At the time of last follow-up, 5 (38.5%) patients had died of disease and 8 patients (61.5%) were alive with no evidence of disease. expression of ezrin and MTA was not significantly different according to the follow-up data. CONCLUSIONS: In our study, high-grade tumors had a higher rate of ezrin and MTA expression. This expression pattern indicates that ezrin and MTA positivity can be additional prognostic markers in osteosarcoma of the jaw.

The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that beta4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of beta4 integrin. Suppression of beta4 integrin expression by shRNA and disruption of beta4 integrin function by transfection of dominant-negative beta4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for beta4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between beta4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The beta4 integrin-ezrin interaction appears to be critical for maintenance of beta4 integrin expression. These data begin to integrate ezrin and beta4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.

The survival of osteosarcoma patients is connected to metastasis. The ezrin expression is associated with the development of metastasis and poor outcome in osteosarcoma. Ezrin is present in the cytoplasm and after phosphorylation assumes an active form and links F-actin to the cell membrane. This study evaluated ezrin and phosphorylated ezrin at site Tyr354 and Thr567 expression and its subcellular localization in osteosarcoma. We studied 50 osteosarcoma patients (mean follow-up 9.8 years). Ezrin expression was assessed using immunohistochemical and immunofluorescence analysis on tissue microarray and cultured cells of human osteosarcoma 143B. The western blot analysis was carried out on cultured cells. The majority of osteosarcomas, showing cytoplasmic positivity for ezrin, phosphorylated and unphosphorylated, were associated with membranous and nuclear positivity for phosphorylated ezrin Thr567 and phosphorylated ezrin Tyr354, respectively. Ezrin expression was associated with high-grade osteosarcoma (P=0.04), with metastasis (P=0.04) and with tumors that developed metastasis (P=0.04); phosphorylated ezrin Thr567 expression was present mostly in tumors with metastasis (P=0.01) and in osteosarcomas that did not develop metastasis (P=0.002). The osteosarcoma patients with ezrin expression have a short survival. The cytoplasmic ezrin expression in osteosarcoma matches its role of membrane-cytoskeleton linker protein. The subcellular trafficking of ezrin is not blocked and it is linked to ezrin phosphorylation, also in cancer. The phosphorylated ezrin Tyr354 nuclear localization suggests its possible role as a nuclear factor in osteosarcoma. The phosphorylated ezrin Thr567 phosphorylation may not be necessary in osteosarcoma metastatic progression but it was modulated. The ezrin expression is associated with more aggressive osteosarcomas and with metastasis.

Osteosarcoma is a life-threatening malignancy that most often occurs in teenagers. Ezrin is a cytoskeleton linker protein that is actively involved in the metastatic capacity of cancer cells. In this study, we investigated the expression change of ezrin after preoperative chemotherapy and its prognostic value in patients with primary osteosarcoma. Ezrin mRNA expression level of initial biopsy specimens and resected tumor specimens after preoperative chemotherapy of 25 patients who had primary osteosarcoma with ezrin expression was detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) to evaluate the expression change of ezrin. In addition, the prognostic factors and the relationship between the expression change of ezrin and the clinical characteristics were analyzed. expression change of ezrin was found in 64% of all 25 patients. The expression change of ezrin had good relation with histology (P = 0.037), grade (P = 0.006), chemotherapy response (P = 0.017), and metastasis or recurrence (P = 0.041). The 2-year overall survival and event-free survival were associated with expression change of ezrin (P = 0.001 and P = 0.002, respectively) and response to preoperative chemotherapy (P = 0.001 and P = 0.012, respectively). Our findings suggest that expression change of ezrin is an independent positive prognostic factor in non-metastatic osteosarcoma.

The cause of death for the vast majority of cancer patients is the development of metastases at sites distant from that of the primary tumor. For most pediatric sarcoma patients such as those with osteosarcoma (OS), despite successful management of the primary tumor through multimodality approaches, the development of metastases, commonly to the lungs, is the cause of death. Significant improvements in long-term outcome for these patients have not been seen in more than 30 years. Furthermore, the long-term outcome for patients who present with metastatic disease is grave [1-5]. New treatment options are needed.Opportunities to improve outcomes for patients who present with metastases and those at-risk for progression and metastasis require an improved understanding of cancer progression and metastasis. With this goal in mind we and others have identified ezrin as a metastasis-associated protein that associated with OS and other cancers. Ezrin is the prototypical ERM (Ezrin/Radixin/Moesin) protein family member. ERMs function as linker proteins connecting the actin cytoskeleton and the plasma membrane. Since our initial identification of ezrin in pediatric sarcoma, an increasing understanding the role of ezrin in metastasis has emerged. Briefly, ezrin appears to allow metastatic cells to overcome a number of stresses experienced during the metastatic cascade, most notably the stress experienced as cells interact with the microenvironment of the secondary site. Cells must rapidly adapt to this environment in order to survive. Evidence now suggests a connection between ezrin expression and a variety of mechanisms linked to this important cellular adaptation including the ability of metastatic cells to initiate the translation of new proteins and to allow the efficient generation of ATP through a variety of sources. This understanding of the role of ezrin in the biology of metastasis is now sufficient to consider ezrin as an important therapeutic target in osteosarcoma patients. This chapter reviews our understanding of ezrin and the related ERM proteins in normal tissues and physiology, summarizes the expression of ezrin in human cancers and associations with clinical parameters of disease progression, reviews reports that detail a biological understanding of ezrin's role in metastatic progression, and concludes with a rationale that may be considered to target ezrin and ezrin biology in osteosarcoma.

OBJECTIVE: to screen microRNA (miRNA) that inhibit expression of the metastasis-related gene ezrin in ovarian cancer cells and explore their correlation to the invasion and metastasis of ovarian cancer. METHODS: the differential expression of ezrin in two paired high-metastatic and low-metastatic cell lines were examined by real time reverse transcription (RT)-PCR and western blot. A functional screen with microarray was employed to identify miRNA that were differentially expressed between SKOV3 and SKOV3ip cell lines. Three programs, TARGETSCAN (http://www.targetscan.org), MICROCOSM (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/) and PICTAR (http://www.pictar.mdc-berlin.de), were employed to identify all miRNA, which may inhibit the expression of ezrin and were differentially expressed between SKOV3 and SKOV3ip cells. To test the repressive potential of these miRNA, synthetic mimetics were transfected individually into SKOV3ip cells and endogenous ezrin expression levels monitored by western blot and real-time RT-PCR. RESULTS: (1) the mRNA average level of ezrin were (81.74 +/- 5.34)-fold higher expression level in SKOV3ip versus SKOV3 cells (P < 0.01), while (2.61 +/- 0.14)-fold in HO-8910PM versus HO-8910 cells (P < 0.01). Elevated protein level of ezrin were observed in SKOV3ip cells compared with that in SKOV3 cells, and the same that in HO-8910PM cells compared with HO-8910 cells. Paired SKOV3 and SKOV3ip cells were employed to study the more significant difference in ezrin expression between them. (2) By a functional screen using miRNA microarray combined with bioinformatics analysis, the miR-183 and miR-22 were indentified as two candidate miRNA, which may have the potential regulatory role in ezrin expression. Real time RT-PCR assays revealed that miR-183 and miR-22 were, respectively, an average of (5.84 +/- 0.66)-fold and (6.67 +/- 0.67)-fold higher expression level in SKOV3ip versus SKOV3 cells (P < 0.01), which were in agreement with the microarray data. A subsequent validation by western blot and real time RT-PCR revealed that over-expression of miR-183 and miR-22 could both lead to an obvious decrease in ezrin protein level, while there were not signicant difference in the level of ezrin mRNA (P > 0.05). CONCLUSION: increased expression of miR-183 and miR-22 may both repress the protein level of ezrin, indicating that miR-183 and miR-22 may bear a potential role in inhibiting ovarian cancer metastasis in a ezrin-mediated way.

BACKGROUND & OBJECTIVE: Ezrin protein is a membrane cytoskeletal crosslinker between the cell membrane and cytoskeleton. Increasing evidence has shown that Ezrin may be associated with tumor invasion and progression. This study was to explore the correlation of Ezrin to metastasis of pancreatic carcinoma. METHODS: The expression of Ezrin mRNA in fresh specimens of 32 pancreatic carcinoma, 19 metastatic lesions and 10 non-cancerous pancreatic tissues were assessed by reverse transcription-polymerase chain (RT-PCR). RESULTS: The mRNA expression of Ezrin was significantly higher in pancreatic carcinoma tissues (1.9+/-1.1) than in non-cancerous tissues (0.9+/-0.5)(P<0.05), significantly higher in metastatic lesions (2.6+/-0.8) than in the primary tumor focus (1.9+/-1.1) (P<0.05), and significantly higher in pancreatic carcinoma lesions with metastasis (2.1+/-1.2) than in those without (1.4+/-0.7) ( P<0.05). Ezrin mRNA was differently expressed in tumors with different clinical pathologic characters. It was highly expressed in poorly differentiated or distantly metastatic carcinomas. CONCLUSIONS: Ezrin gene is closely related to invasion and metastasis of pancreatic carcinoma, which might be used as a marker in predicting the metastasis of pancreatic carcinoma.

The aim of the study was to observe different protein profiles in pancreatic cancer with and without lymph node metastasis (LNM), and search for novel LNM-associated proteins, which would help to understand the metastatic mechanisms and provide targets for therapeutic interventions. cancer nests were manually miscrodissected from 8 LNM and 7 non-LNM pancreatic cancer tissues, and the protein extracts were then separated by difference gel electrophoresis (DIGE) and identified by MALDI-TOF-TOF. Four differently regulated proteins, ezrin, radixin, moesin, and c14orf166, were selected for further validation by Western blot and immunohistochemistry. In DIGE analysis, we identified 18 up-regulated proteins and 15 down-regulated proteins in LNM pancreatic cancer nests compared with non-LNM ones. Western blot and immunohistochemical analyses confirmed that radixin, moesin and c14orf166, but not ezrin, had significantly higher expression levels in LNM pancreatic cancers than in non-LNM controls. In conclusion, the specific protein profiles found in this study might provide new insights into the mechanism of lymph node metastasis. For the first time, c14orf166 was identified asa novel metastasis-associated protein, and the roles of radixin, moesin and c14orf166 in cancer metastasis deserve further investigations.

Patients presenting with metastatic rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children, have a very poor clinical prognosis. This is due, in large part, to our rudimentary knowledge of the molecular events that dictate metastatic potential. We used cDNA microarray analysis of RMS cell lines, derived from Ink4a/Arf-deficient mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF), to identify a set of genes whose expression was significantly different between highly and poorly metastatic cells. Subsequent in vivo functional studies revealed that the actin filament-plasma membrane linker ezrin (encoded by Vil2) and the homeodomain-containing transcription factor Six-1 (sine oculis-related homeobox-1 homolog) had essential roles in determining the metastatic fate of RMS cells. VIL2 and SIX1 expression was enhanced in human RMS tissue, significantly correlating with clinical stage. The identification of ezrin and Six-1 as critical regulators of metastasis in RMS provides new mechanistic and therapeutic insights into this pediatric cancer.

PURPOSE: Ezrin is a cytoskeleton linker protein that is actively involved in regulating the growth and metastatic capacity of cancer cells. It has recently been reported to be involved in dissemination of pediatric soft tissue sarcoma (STS). EXPERIMENTAL DESIGN: To further evaluate the prognostic value of ezrin in STS progression, we screened 50 primary STSs of high malignancy grade using immunohistochemistry. At the initial surgery, all patients were without local or distant metastasis. The expression was then compared with the outcome during follow-up for at least 4 years or until the patients' death. RESULTS: Twenty-five of the 50 STSs analyzed (50%) showed ezrin immunoreactivity in the membrane and cytoplasm of the tumor cells. A significant association was shown between positive expressions of ezrin and death in disease as well as overall survival (P = 0.014 and 0.007, respectively). Similarly, ezrin expression was significantly associated with development of distant metastasis during follow-up (P = 0.031), also excluding locally recurrent disease (P = 0.049). The relative abundance of metastasis in ezrin-positive cases was observed both over time and irrespective of time. In comparison with clinical, histopathologic, and genetic characteristics of the STSs, ezrin expression was found to correlate significantly with an infiltrative growth pattern outside the tumor capsule as well as with copy number gain of chromosomal region 9cen-q22. CONCLUSION: Our findings suggest that ezrin immunoreactivity could be valuable as an additional prognostic marker in highly malignant STSs and support a causative role of ezrin in STS tumor dissemination.

OBJECTIVE: To investigate the effects of Ezrin, Moesin and E-Cadherin on the invasion and metastasis of papillary thyroid carcinoma(PTC). METHODS: The 81 paraffin-embedded specimen of PTC treated in The Second Clinical College of Harbin Medical University from 2005 to 2008 were examined immunohistochemically for Ezrin, Moesin, and E-Cadherin. Normal thyroid tissues adjacent to cancer were control. The correlation of Ezrin, Moesin, and E-Cadherin expressions and clinical features of PTC, including gender, age, tumor size, invasion, lymph node metastasis, and TNM stage, was analyzed. RESULTS: Positive staining rates of Ezrin and Moesin in PTC were significantly higher than those in control (chi(2) were 61.691 and 57.949, P < 0.01, respectively). Positive staining rate of E-Cadherin in PTC was significantly lower than that in control (chi(2) = 64.241, P < 0.01). The positive expression rates of Ezrin and Moesin in PTC increased in the subgroups with age >/= 45 years, local invasion, lymph node metastasis and stage III-IV, with significant difference respectively (P < 0.01), while the positive expression rate of E-Cadherin in PTC significantly decreased (P < 0.01). CONCLUSIONS: The up-regulation expressions of Ezrin and Moesin and the down-regulation expression of E-Cadherin were associated with the invasion and metastasis of PTC. Combined detection of Ezrin, Moesin and E-Cadherin in PTC might served as an important predictor for the invasion, metastasis and prognosis of PTC.

OBJECTIVE: In order to assess the significance of Ezrin and E-Cadherin in invasion and metastasis of papillary thyroid carcinoma (PTC). METHOD: The 81 paraffin-embedded specimen PTC and normal thyroid tissues adjacent to cancer operated from 2005 to 2008 were tested. The expressions of Ezrin, E-Cadherin in the specimen were assayed by immunohistochemical staining. The correlation between the 2 factors (Ezrin, E-Cadherin) and clinical features of PTC such as gender, age, tumor size, invasion, lymph node metastasis, and TNM stage were studied. RESULTS: The rates of immuno-positive staining Ezrin in PTC was higher than the normal thyroid tissues adjacent to cancer with significant difference respectively (chi2 = 61.691, P < 0.01). The rates of immuno-positive staining E-Cadherin in PTC was lower than the normal thyroid tissues adjacent to cancer with significant difference respectively (chi2 = 64.241, P < 0.01). The expressions of Ezrin and E-cadherin were related to age, invasion, lymph node metastasis, and TNM. There was correlation between the expressions of Ezrin and E-Cadherin in PTC (r = -0.471, P < 0.01). CONCLUSION: The high expressions of Ezrin and the low expression of E-Cadherin were related to invasion and metastasis in PTC. Between the 2 factors has cooperative. Combined detection of the expression of Ezrin and E-Cadherin in PTC might served as invasion, metastasis and important prognostic predictors.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a crucial programme in cancer metastasis. Epidermal growth factor (EGF) is a key inducer of EMT, and Ezrin has an important role in this process. However, how Ezrin is activated and whether it mediates EGF-induced EMT in tongue squamous cell carcinomas (TSCCs) through activating NF-kappaB remains obscure. METHODS: We used two TSCC cell lines as a cell model to study invasion and EMT in vitro, and used nude mice xenografts model to evaluate metastasis of TSCC cells. Finally, we evaluated the level of pEzrin Tyr353, nuclear p65 and EMT markers in TSCC clinical samples. RESULTS: Ezrin Tyr353 was phosphorylated through Akt (but not ERK1/2, ROCK1) pathway, and lead to the activation of NF-kappaB in EGF-treated TSCC cells. Akt and NF-kappaB inhibitors blocked EGF-induced EMT, and suppressed invasion and migration of TSCC cells. In vivo, silencing Ezrin significantly suppressed EGF-enhanced metastasis of TSCC xenografts. Finally, high levels of expression of pEzrin Tyr353, nuclear p65, vimentin and low level of expression of E-cadherin were correlated with cancer metastasis and poor patient prognosis. CONCLUSION: Our data suggest that Akt/Ezrin Tyr353/NF-kappaB pathway regulates EGF-induced EMT and metastasis inTSCC, and Ezrin may serve as a therapeutic target to reverse EMT in tongue cancers and prevent TSCC progression.