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Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation. The N-BAR domain protein BIN1 acts as a scaffolding protein during filopodia formation in myoblast cells, together with identified co-factors. [ABSTRACT FROM AUTHOR]

CLIC5 belongs to a family of ion channels with six members reported so far. In vertebrates, the CLIC5 gene encodes two different isoforms, CLIC5A and CLIC5B. In addition to its ion channel activity, there is evidence for further functions of CLIC5A, such as the remodeling of the actin cytoskeleton during the formation of a functional glomerulus in the vertebrate kidney. However, its specific role is still incompletely understood and a specific functional role for CLIC5B has not been described yet. Here we report our findings on the differential expression and functions of Clic5a and Clic5b during zebrafish kidney development. Whole-mount in situ hybridization studies revealed specific expression of clic5a in the eye and pronephric glomerulus, and clic5b is expressed in the gut, liver and the pronephric tubules. Clic5 immunostainings revealed that Clic5b is localized in the cilia. Whereas knockdown of Clic5a resulted in leakiness of the glomerular filtration barrier, Clic5b deficient embryos displayed defective ciliogenesis, leading to ciliopathy-associated phenotypes such as ventral body curvature, otolith deposition defects, altered left–right asymmetry and formation of hydrocephalus and pronephric cysts. In addition, Clic5 deficiency resulted in dysregulation of cilia-dependent Wnt signalling pathway components. Mechanistically, we identified a Clic5-dependent activation of the membrane-cytoskeletal linker proteins Ezrin/Radixin/Moesin (ERM) in the pronephric tubules of zebrafish. In conclusion, our in vivo data demonstrates a novel role for Clic5 in regulating essential ciliary functions and identified Clic5 as a positive regulator of ERM phosphorylation. [ABSTRACT FROM AUTHOR]

Background: Ezrin, known as a crosslinker between the plasma membrane and actin cytoskeleton, is closely associated with breast cancer (BC) progression. Here, we explored a novel role of ezrin in breast cancer liver metastasis (BCLM). Methods: The clinical relevance of ezrin was evaluated using in silico tools and confirmed in BC specimens. The effect of ezrin on proliferation, migration and invasion was examined in vitro and in vivo using murine primary liver-metastatic breast cancer cells (mLM). The molecular mechanism involved in ezrin-mediated activation of the Notch1 signaling pathway was elucidated using in vitro models. Results: Data-mining demonstrated that ezrin mRNA and protein expression is up-regulated in breast cancer cohorts and has prognostic significance. Ezrin overexpression promotes cell proliferation, migration and invasion in vitro and in vivo. Hairy and enhancer of split-1 (Hes1) is one of the most significantly enriched candidates of differentially expressed genes in ezrin overexpression and control mLM cells. Ezrin can positively regulate Hes1 mRNA and protein expression, and their coexpression was associated with poor prognosis in BC patients. Ezrin promoted BC cell proliferation in a Hes1-dependent manner without directly interacting with Hes1. The functional link between ezrin and Hes1 is dependent on Notch1 activation through promotion of furin-like convertase cleavage. Conclusion: Our results demonstrated that ezrin drives BCLM through activation of the Notch signaling pathway via furin-like convertase. These findings provide a better understanding of the mechanism of ezrin in breast cancer progression, with the goal of discovering a novel target for the treatment of BCLM in the future. [ABSTRACT FROM AUTHOR]

Recurrence and progression rates vary widely among different histological subtypes of bladder cancer (BC). Normal-appearing mucosa in non-muscle invasive bladder cancer and muscle-invasive bladder cancer in cystoscopy and histopathology is a factor in staging and treatment. Telocytes (TCs) are spindle-shaped cells that connect with other cell types allowing communication though cytoskeletal signaling. They are involved in tissue regeneration and pathogenesis of diseases and cancer. In this study, 12 normal-appearing tissues from urinary bladder with BC, both invasive and non-invasive were evaluated in patients who had either trans-urethral resection of bladder tumor or cystectomy. In each case, cystoscopy, intraoperative inspection, and histopathology all confirmed the absence of cancerous elements. Five patients with neurogenic bladder were used as a control group. Immunohistochemistry revealed that c-Kit + cells were intensively distributed in bladder layers from BC samples, while they were seldom detected in the control group. Ultrastructural examination of reprocessed tissue showed an intense distribution of TCs and telopodes in the submucosa and between smooth muscle cells in BC. Telopodes were numerous, arborizing, and intercommunicating. Whereas TCs and telopodes were scarce in the neurogenic bladder. Also, cancerous tissue had the highest expression level of ezrin protein, and this level gradually decreased as we moved away from the tumor. Our finding of TCs number in normal-appearing tissues in conjunction with ezrin expression may compete invasiveness and possibly a trail to reduce recurrence rates. [ABSTRACT FROM AUTHOR]

Discovering novel changes in the proteome of malignant lung epithelial cells and/or the tumor-microenvironment is paramount for diagnostic, prognostic, and/or therapy development. A time-dependent 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumor model was used to screen the proteome of lung tumors. NNK-transformed human lung epithelial BEAS-2B cells were then established to evaluate the epithelial cell-specific protein changes. A duration-dependent increase of tumor burden was observed in NNK-treated mice, 2/12 (17%), 8/12 (67%), 9/12 (75%), and 10/10 (100%) at weeks 8, 12, 16, and 20 after the NNK exposure, respectively. A total of 25 differentially expressed proteins (≥ twofold change), predominantly structural, signaling, and metabolic proteins, were detected by two-dimensional difference gel electrophoresis and identified by mass spectrometry. Calregulin, ezrin, histamine releasing factor (HRF), and inorganic pyrophosphatase 1 (PPA1) exhibited changes and were further confirmed via immunoblotting. In addition, immunohistochemistry (IHC) analysis indicated upregulated E-cadherin and decreased vimentin expression in epithelial cells of tumor tissues. Acquisition of a neoplastic phenotype in NNK-transformed BEAS-2B cells was demonstrated by enhanced wound closure and increased anchorage independent colony formation. In transformed BEAS-2B cells, protein expression of E-cadherin, ezrin, and PPA1 (but not calregulin and HRF) was upregulated, as was observed in tumor tissues IHC staining using mouse lung tumor tissues further revealed that HRF upregulation was not lung epithelial cell specific. Altogether, tumorigenesis after NNK exposure may be initiated by protein dysregulation in lung epithelial cells together with proteome derangement derived from other cell types existing in the tumor-microenvironment. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN) is intensely expressed on the podocyte membrane in an evolutionally conserved manner. CLEC-2, the endogenous ligand of PDPN, is highly expressed in platelets and also exists in a soluble form in plasma. Normally, podocytes are sequestered from CLEC-2, but when the glomerular barrier is injured, podocytes gain access to CLEC-2. We tested the effects of CLEC-2 in podocytes in vitro and in vivo. Cultured podocytes treated with Fc-CLEC-2 demonstrated that CLEC-2 induced the dephosphorylation of ezrin, radixin, and moesin (ERM) proteins. Podocytes treated with Fc-CLEC-2 also showed the dissociation of F-actin filaments from PDPN, F-actin degradation, detachment, and round morphology. Next, we perfused normal mouse kidney in vivo with FLAG-CLEC-2. CLEC-2 induced dephosphorylation of ERM and widening of the foot processes of podocytes. Platelets were detected by immunostaining for CD41 in the urine of mice with podocyte injury, indicating that podocytes can encounter platelets when glomeruli are injured. Collectively, these observations suggest that when platelets leak through the injured glomeruli, CLEC-2 from the platelets acts on PDPN in podocytes and induces morphological change and detachment, which may further aggravate podocyte injury. Thus, PDPN on podocytes may work as a leaked-platelet sensor. [ABSTRACT FROM AUTHOR]

Placental dysplasia increases the risk of recurrent spontaneous abortion (RSA). However, the underlying mechanism regulating placental development remains unclear. In this study, we showed that the expression of CDC42 was decreased in the villous tissue of RSA samples compared to healthy controls. Further examination demonstrated that CDC42 deficiency led to the differentiation of human trophoblast stem cells (hTSCs) and inhibited their proliferation. Genetic manipulation of YAP and EZRIN in hTSCs revealed that CDC42 regulates the stemness and proliferation of hTSCs; this is dependent on EZRIN, which translocates YAP into the nucleus. Moreover, the expression pattern of EZRIN, YAP, and Ki67 was also abnormal in the villous tissue of RSA samples, consistent with in vitro experiments. In summary, these findings suggest that the CDC42/EZRIN/YAP pathway plays an important role in placental development. [ABSTRACT FROM AUTHOR]

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Purpose: Despite great advances that have been made in the understanding of the molecular complexity of acute myeloid leukemia (AML), very little has been translated into new therapies. Here, we set out to investigate the impact of cytoskeleton regulatory genes on clinical outcomes and their potential as therapeutic targets in AML. Methods: Gene expression and clinical data were retrieved from The Cancer Genome Atlas (TCGA) AML study and used for survival and functional genomics analyses. For pharmacological tests, AML cells were exposed to ezrin (EZR) inhibitors and submitted to several cellular and molecular assays. Results: High EZR expression was identified as an independent marker of worse outcomes in AML patients from the TCGA cohort (p < 0.05). Functional genomics analyses suggested that EZR contributes to responses to stimuli and signal transduction pathways in leukemia cells. EZR pharmacological inhibition with NSC305787 and NSC668394 reduced viability, proliferation, autonomous clonal growth, and cell cycle progression in AML cells (p < 0.05). NSC305787 had a greater potency and efficiency than NSC668394 in leukemia models. At the molecular level, EZR inhibitors reduced EZR, S6 ribosomal protein and 4EBP1 phosphorylation, and induced PARP1 cleavage in AML cells. NSC305787, but not NSC668394, favored a gene network involving cell cycle arrest and apoptosis in Kasumi 1 AML cells. Conclusions: From our data we conclude that EZR expression may serve as a prognostic factor in AML. Our preclinical findings indicate that ezrin inhibitors may be employed as a putative novel class of AML targeting drugs. [ABSTRACT FROM AUTHOR]

Kindlin3 (K3), a FERM domain containing protein expressed in hematopoietic cells controls integrin activation and thus hemostatic and inflammatory responses. However, its role in the mechanics of plasma membrane remains unclear. Here, we show that genetic knockout of K3 in microglia and macrophages resulted in defective plasma membrane tension and membrane blebbing. Atomic force microscopy (AFM) of K3-deficient cells revealed a significant loss in membrane-to-cortex attachment (MCA), and consequently reduced membrane tension. This loss in MCA is amplified by the mislocalization of the cell cortex proteins—ezrin, radixin, and moesin (ERM)—to the plasma membrane of microglia and macrophages. Re-expression of K3 in K3-deficient macrophages rescued the defects and localization of ERMs implying a key role for K3 in MCA. Analysis of two K3 mutants, K3int affecting integrin binding and activation, and K3pxn/act disrupting binding to paxillin and actin but not integrin functions, demonstrated that the role of K3 in membrane mechanics is separate from integrin activation. The K3pxn/act mutant substantially diminished both membrane tension and Yes-associated protein (YAP) translocation to the nucleus, while preserving integrin activation, cell spreading, and migration. Together, our results show that K3 coordinates membrane mechanics, ERM protein recruitment to the membrane, and YAP translocation by linking integrin at the membrane to paxillin and actin of the cytoskeleton. This novel function of K3 is distinct from its role in integrin activation. [ABSTRACT FROM AUTHOR]

The actin-binding proteins profilin, fascin, and ezrin were tested for involvement in metastasis of non-small cell lung cancer (NSCLC). The levels of the PFN1, FSCN1, and EZR mRNAs and respective proteins were determined by real-time PCR and Western blotting; tumor and adjacent normal lung tissue samples were obtained from 46 NSCLC patients. Patients with lymphatic metastasis had higher expression levels of the profilin, fascin, and ezrin mRNAs and the profilin and fascin proteins. Both mRNA and protein expression levels increased in patients with distant metastasis. The molecules may serve as predictors to evaluate the prognosis in NSCLC. [ABSTRACT FROM AUTHOR]

Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination. [ABSTRACT FROM AUTHOR]

Purpose: Triple negative breast cancer (TNBC) patients generally have an adverse clinical outcome because their tumors often recur and metastasize to distant sites in the first 3 years after surgery. Therefore, it has become pivotal to identify potential factors associated with metastasis. Here, we focused on the effects of S100P and Ezrin on the trans-endothelial migration (TEM) of TNBC cells, as they have both been suggested to play a role in this process in other malignancies.Methods: The expression of S100P and Ezrin was examined by immunohistochemistry in 58 primary TNBC samples. The mRNA and protein levels of S100P and Ezrin were assessed in breast cancer-derived cell lines using qRT-PCR and Western blotting, respectively. Proliferation and migration assays were performed using TNBC-derived MFM-223 and SUM-185-PE cells transfected with S100P and Ezrin siRNAs. Two different timeframes were employed for TEM assays using TNBC-derived cells and human umbilical vein endothelial-derived cells, respectively. Correlations between the status of EzrinThr-567 expression and various clinicopathological features were analyzed by immunohistochemistry.Results: We found that S100P and Ezrin double negative TNBC cases were significantly associated with a better disease-free survival. We also found that single and double siRNA-mediated knockdown of S100P and Ezrin in TNBC-derived cells significantly inhibited their TEM and destabilized the intercellular junctions of endothelial cells. In addition, we found that EzrinThr-567 immunoreactivity significantly correlated with vascular invasion in TNBC patients.Conclusions: From our data we conclude that S100P, Ezrin and EzrinThr-567 are involved in the trans-endothelial migration of TNBC cells and that they may serve as potential targets in TNBC patients. [ABSTRACT FROM AUTHOR]

Objectives: Microvillus inclusion disease (MVID) is a severe form of neonatal diarrhea, caused mainly by mutations in MYO5B. Inactivating mutations in MYO5B causes depolarization of enterocytes in the small intestine, which gives rise to chronic, unremitting secretory diarrhea. While the pathology of the small intestine in MVID patients is well described, little is known about extraintestinal effects of MYO5B mutation.Methods: We examined stomach, liver, pancreas, colon, and kidney in Navajo MVID patients, who share a single homozygous MYO5B-P660L (1979C>T p.Pro660Leu, exon 16). Sections were stained for markers of the apical membrane to assess polarized trafficking.Results: Navajo MVID patients showed notable changes in H/K-ATPase-containing tubulovesicle structure in the stomach parietal cells. Colonic mucosa was morphologically normal, but did show losses in apical ezrin and Syntaxin 3. Hepatocytes in the MVID patients displayed aberrant canalicular expression of the essential transporters MRP2 and BSEP. The pancreas showed small fragmented islets and a decrease in apical ezrin in pancreatic ducts. Kidney showed normal primary cilia.Conclusions: These findings indicate that the effects of the P660L mutation in MYO5B in Navajo MVID patients are not limited to the small intestine, but that certain tissues may be able to compensate functionally for alterations in apical trafficking. [ABSTRACT FROM AUTHOR]

L1 cell adhesion molecule (L1CAM) is highly expressed in various types of human cancers, displaying yet unknown molecular mechanisms underlying their oncogenic potential. Here, we found that L1CAM expression was significantly increased in esophageal squamous cell carcinoma (ESCC; n = 157) lesions compared with non-cancerous tissues. High tumorous L1CAM expression significantly correlated with reduced overall survival. Experimentally, L1CAM knockdown led to decreased cell growth, migration, and invasiveness in vitro, whereas overexpression of L1CAM showed the opposite effect. In nude mice, L1CAM depletion attenuated tumorigenesis and ability to penetrate the tissues surrounding ESCC cells. Gene set enrichment analysis (GSEA) and SubpathwayMiner analysis on gene expression profiles (microarray data on ESCC tissues, GSE53625; cDNA microarray data on L1CAM-knockdown ESCC cell line, GSE86268) suggested that L1CAM-co-expression genes were related to cell motility, cell proliferation, and regulation of actin cytoskeleton, validating the above experimental findings. Further mechanistical analysis showed that L1CAM upregulated the expression of the cytoskeletal protein ezrin via activating integrin β1/MAPK/ERK/AP1 signaling and thus led to the malignant phenotypes of ESCC cells. Together, our findings suggest that L1CAM may be employed as a valuable prognosis marker and a therapeutic target for ESCC patients and that L1CAM promotes ESCC tumorigenicity by upregulating ezrin expression. Key messages: [ABSTRACT FROM AUTHOR]

Introduction: Podoplanin and ezrin connection through Rho-A phosphorylation have been suggested as part of the activation pathway, in the process of tumor invasion and cell movement in oral squamous cell carcinomas. Objective: The aim of this study was to evaluate the correlation among podoplanin, ezrin, and Rho-A immunoexpressions in 91 squamous cells carcinomas of the lower lip and their influence in patient's prognosis. Material and methods: The immunoexpressions of podoplanin, ezrin, and Rho-A were evaluated through a semi-quantitative score method, based on the capture of 10 microscopic fields at the front of tumor invasion. The association and correlation of these proteins with the clinicopathological features were verified by Fischer's exact test and Spearman's test. The prognostic values were analyzed by Kaplan-Meier method and log-rank test. Results: A statistically significant association between strong cytoplasmic podoplanin expression and alcohol ( p = 0.024), loco-regional recurrences ( p = 0.028), and lymph node metastasis (pN+) ( p = 0.010) was found. The membranous ( p = 0.000 and r = 0.384) and cytoplasmic ( p = 0.000 and r = 0.344) podoplanin expression was statistically correlated with ezrin expression. Also, membranous podoplanin was significantly correlated with Rho-A expression ( p = 0.006 and r = 0.282). The expressions of podoplanin, ezrin, and Rho-A were not significant prognostic factors for patients with squamous cell carcinomas of the lower lip. Conclusions: Therefore, our results confirm a correlation among podoplanin, ezrin, and Rho-A expressions in squamous cell carcinoma of the lip suggesting a cooperative participation of these proteins in cell movement and invasion. Clinical relevance: Furthermore, strong cytoplasmic podoplanin expression could be helpful to identify patients with squamous cell carcinoma of the lip and lower risk of loco-regional recurrences. [ABSTRACT FROM AUTHOR]

Rationale: Development of cervical squamous carcinoma (CXCA) is accompanied by changes in estrogen receptors (ERs, ERα and ERβ) and ezrin expression; however, reports have been conflicting. Using histologically documented staging of cervical biopsies, we determined ezrin and ER relationships during CXCA development. Methods: Immunoreactive (ir) ezrin, ir-ERα, and ir-ERβ were studied in normal epithelium, carcinoma in situ/cervical intraepithelial neoplasia (CIN) 1 to 3, and local invasion or metastatic CXCA. Results were compared using H scoring. Cultures of Caski metastatic CXCA cells were treated with estradiol and/or tamoxifen and studied for ER-driven ir-ezrin and the morphologic response. Results: Koilocytosis was present and indicated viral presence. The ezrin H score increased from CIN1 to CIN3, reaching significant differences from normal by CIN3 (P = .004) and 2× normal in metastatic CXCA. Estrogen receptor α and ERβ H scores fell, reaching significance by CIN3 (ERα, P = .0001; ERβ, P = .024). During estradiol treatment, ezrin in Caski cells increased and localized to the periphery, in ruffles and processes. The selective ER modulator tamoxifen blocked the estradiol-induced changes. Conclusions: During cervical carcinogenesis, the usual relationship between estrogen and ezrin induction is abridged. This is consistent with the effects of human papilloma virus viral proteins such as E6 and E7 that upregulate SIX1, a protein that induces ezrin. Cervical carcinogenesis is progressive but arrests at the preinvasive stage for varying lengths of time. These studies suggest that changes in ezrin may be associated with the development of the invasive phenotype and penetration of the basement membrane. They also raise the possibility that inhibiting ezrin expression could be a target for the prevention of invasive CXCA. [ABSTRACT FROM AUTHOR]

Purpose: Histologic classification of invasive lung adenocarcinomas by predominant subtype has prognostic value. Papillary predominant adenocarcinoma (PPA) reportedly shows poorer prognosis than lepidic predominant adenocarcinoma (LPA); however, biological differences between PPA and LPA are unclear. The purpose of this study was to clarify biological differences between PPA and LPA. Methods: Clinicopathological characteristics of invasive 62 PPAs and 117 LPAs smaller than 30 mm were investigated. Furthermore, we compared immunochemical staining scores of 9 molecular markers (E-cadherin, S100A4, fibronectin, integrinβ1, ezrin, GLUT1, ALDH1, SOX2 and Nanog) between PPA and LPA. We performed Western blot analysis using ezrin shRNA-knockdown lung adenocarcinoma cell lines to examine whether molecules that are highly expressed in PPA, such as ezrin, affect pAkt. Finally, we performed immunochemical staining to compare pAkt expression level in PPA and LPA. Results: Lymphovascular and pleural invasion and lymph node metastasis were significantly more often detected in PPA than in LPA (lymphatic permeation: 31 vs 3 %, vascular invasion: 35 vs 3 %, pleural invasion: 29 vs 5 %, lymph node metastasis: 18 vs 1 %; all P < 0.01). Immunohistochemical (IHC) study revealed that expression score of ezrin was significantly higher in PPA than in LPA (38.3 vs 15.0; P < 0.01). The level of pAkt decreased in shEzrin-induced PC-9 and A549 cancer cells. Moreover, the IHC staining score of pAkt was significantly higher in PPA than in LPA (13.3 vs 0.0; P < 0.01). Conclusions: Our results show that the activation of the ezrin-pAkt signaling axis is associated with the more aggressive clinicopathological features of PPA compared with LPA. [ABSTRACT FROM AUTHOR]

Purpose: CITED4 is one member of a family of transcriptional cofactors, several of which are deregulated in a variety of tumors, including colorectal cancer (CRC). We modulated CITED4 expression, in vitro, and analyzed the associated phenotypic and gene expression changes. Methods: CITED4-overexpressing and shRNA-mediated knockdown cell lines and control cell lines were established in the CRC cell line SW480. The cells were analyzed for changes in proliferation, apoptosis/cell cycle, migration, invasion, colony formation and adhesion. mRNA expression changes were determined by microarray and pathway analysis, and several deregulated genes were validated by qRT-PCR and Western blotting. Based on results obtained from these studies, the status of the actin cytoskeleton was evaluated by phalloidin/vinculin staining. Results: Phenotypically, the CITED4-overexpressing cell line showed only moderate changes in adhesion. Microarray analysis identified several deregulated genes, including several G protein-coupled receptors. Phenotypic analysis of the CITED4 shRNA knockdown cell line demonstrated decreased cell proliferation and G2 cell cycle blockage. Microarray analysis identified many deregulated genes, and pathway analysis discovered genes linked to actin-associated adherens junctions/tight junctions (claudin-4, claudin-7, ezrin, MET, ß-catenin). Phenotypically, no morphological changes of the actin cytoskeleton were seen. Conclusions: Upregulation of CITED4 in SW480 resulted in no obvious phenotype. CITED4 shRNA-mediated knockdown led to decreased cellular proliferation and modulation of a large number of genes, including the c-MET tyrosine kinase and several actin-associated adherens junctions/tight junction genes. [ABSTRACT FROM AUTHOR]

The peripheral astrocyte process (PAP) is the glial compartment largely handling inactivation of transmitter glutamate, and supplying glutamate to the axon terminal. It is not clear how these energy demanding processes are fueled, and whether the PAP exhibits oxidative capability. Whereas the GFAP-positive perinuclear cytoplasm and stem process are rich in mitochondria, the PAP is often considered too narrow to contain mitochondria and might thus not rely on oxidative metabolism. Applying high resolution light microscopy, we investigate here the presence of mitochondria in the PAPs of freshly dissociated, isolated astrocytes. We provide an overview of the subcellular distribution and the approximate size of astrocytic mitochondria. A substantial proportion of the astrocyte's mitochondria are contained in the PAPs and, on the average, they are smaller there than in the stem processes. The majority of mitochondria in the stem and peripheral processes are surprisingly small (0.2-0.4 µm), spherical and not elongate, or tubular, which is supported by electron microscopy. The density of mitochondria is two to several times lower in the PAPs than in the stem processes. Thus, PAPs do not constitute a mitochondria free glial compartment but contain mitochondria in large numbers. No juxtaposition of mitochondria-containing PAPs and glutamatergic synapses has been reported. However, the issue of sufficient ATP concentrations in perisynaptic PAPs can be seen in the light of (1) the rapid, activity dependent PAP motility, and (2) the recently reported activity-dependent mitochondrial transport and immobilization leading to spatial, subcellular organisation of glutamate uptake and oxidative metabolism. [ABSTRACT FROM AUTHOR]

Plasma membrane tension is responsible for a variety of cellular functions such as motility, cell division, and endocytosis. Since membrane tension is dominated by the attachment of the actin cortex to the inner leaflet of the plasma membrane, we investigated the importance of ezrin, a major cross-linker of the membrane-cytoskeleton interface, for cellular mechanics of confluent MDCK II cells. For this purpose, we carried out ezrin depletion experiments and also enhanced the number of active ezrin molecules at the interface. Mechanical properties were assessed by force indentation experiments followed by membrane tether extraction. PIP2 micelles were injected into individual living cells to reinforce the linkage between plasma membrane and actin-cortex, while weakening of this connection was reached by ezrin siRNA and administration of the inhibitors neomycin and NSC 668394, respectively. We observed substantial stiffening of cells and an increase in membrane tension after addition of PIP2 micelles. In contrast, reduction of active ezrin led to a decrease of membrane tension accompanied by loss of excess surface area, increase in cortical tension, remodelling of actin cytoskeleton, and reduction of cell height. The data confirm the importance of the ezrin-mediated connection between plasma membrane and cortex for cellular mechanics and cell morphology. [ABSTRACT FROM AUTHOR]

Podoplanin was identified as a protein associated with the transformation of arborized foot processes of glomerular epithelial cells (podocytes) to flat feet. However, the function of podoplanin in the podocyte is not yet fully clarified. In this study, we analyzed the molecular nature of podoplanin, and its expression in rat nephrotic models and patients with minimal change nephrotic syndrome (MCNS). We demonstrated here that podoplanin has two forms: one contains abundant sialic acid and the other a lesser amount of sialic acid. Podoplanin bound ezrin to interact with the cytoskeleton. The silencing of podoplanin in cultured podocytes caused a change in the cell shape and the distribution of ezrin and actin. The expression of podoplanin was clearly reduced before the onset of proteinuria in puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, and the decrease in the expression of podoplanin became more evident at the proteinuric stage. Podoplanin was detected in normal urine samples, and the amount of urinary podoplanin markedly increased on day 1 of PAN nephropathy. Urinary ezrin was also detected. The amount of the phosphorylated ezrin was reduced, while the amount of the podoplanin-interacting ezrin increased. The podoplanin expression was reduced in a patient with active-phase MCNS. It is conceivable that the alteration of the podoplanin-ezrin-cytoskeleton linkage is an important event of the podocyte injury in MCNS. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is a hematological cancer associated with an aggressive clinical course. The predominant subtypes of DLBCL display features of chronic or tonic B-cell antigen receptor (BCR) signaling. However, it is not known whether the spatial organization of the BCR contributes to the regulation of pro-survival signaling pathways and cell growth. Here, we show that primary DLBCL tumors and patient-derived DLBCL cell lines contain high levels of phosphorylated Ezrin-Radixin-Moesin (ERM) proteins. The surface BCRs in both activated B cell and germinal B cell subtype DLBCL cells co-segregate with phosphoERM suggesting that the cytoskeletal network may support localized BCR signaling and contribute to pathogenesis. Indeed, ablation of membrane-cytoskeletal linkages by dominant-negative mutants, pharmacological inhibition and knockdown of ERM proteins disrupted cell surface BCR organization, inhibited proximal and distal BCR signaling, and reduced the growth of DLBCL cell lines. In vivo administration of the ezrin inhibitor retarded the growth of DLBCL tumor xenografts, concomitant with reduction in intratumor phosphoERM levels, dampened pro-survival signaling and induction of apoptosis. Our results reveal a novel ERM-based spatial mechanism that is coopted by DLBCL cells to sustain tumor cell growth and survival. [ABSTRACT FROM AUTHOR]

Purpose: Cancer cells and cancer-associated fibroblasts (CAFs) together create the tumor microenvironment, which affects malignant behavior. Lung adenocarcinomas with CAFs expressing podoplanin (PDPN) are clinically aggressive, but the molecular mechanism underlying this phenomenon has not been established. So we identified the characteristic immunophenotype of lung adenocarcinoma cells coexisting with PDPN-expressing CAFs (PDPN-CAFs) and examined how it relates to an aggressive clinicopathological outcome. Methods: We analyzed the clinicopathological characteristics of 119 adenocarcinomas with a uniform size (2-3 cm). The expression levels of ten invasiveness-related proteins which related to cell adhesion and invasiveness, such as Ezrin, were examined in cancer cells from PDPN-CAFs (+) cases and from PDPN-CAFs (−) cases ( n = 20 each). To examine the functional importance of the identified protein on the invasion phenotype, we performed wound healing and a Matrigel invasion assay using shRNA-knockdown lung adenocarcinoma cells (PC-9). Results: The PDPN-CAFs (+) cases had significantly higher rates of node metastasis ( p < 0.01) and vascular invasion ( p < 0.01). The cancer cells from the PDPN-CAFs (+) cases also had a significantly higher staining score for Ezrin ( p < 0.01) than those from the PDPN-CAFs (−) cases. The migration and invasion activities of the shEzrin-induced PC-9 cells were significantly lower than those of the control cells. Conclusions: Our results indicated that within a tumor microenvironment composed of PDPN-CAFs, increased Ezrin expression in cancer cells might play a key role in the invasiveness of lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Hepatitis C virus infection of hepatocytes is a multistep process involving the interaction between viral and host cell molecules. Recently, we identified ezrin-moesin-radixin proteins and spleen tyrosine kinase (SYK) as important host therapeutic targets for HCV treatment development. Previously, an ezrin hinge region peptide (Hep1) has been shown to exert anti-HCV properties in vivo, though its mechanism of action remains limited. In search of potential novel inhibitors of HCV infection and their functional mechanism we analyzed the anti-HCV properties of different human derived radixin peptides. Sixteen different radixin peptides were derived, synthesized and tested. Real-time quantitative PCR, cell toxicity assay, immuno-precipitation/western blot analysis and computational resource for drug discovery software were used for experimental analysis. We found that a human radixin hinge region peptide (Peptide1) can specifically block HCV J6/JFH-1 infection of Huh7.5 cells. Peptide 1 had no cell toxicity or intracellular uptake into Huh7.5 cells. Mechanistically, the anti-HCV activity of Peptide 1 extended to disruption of HCV engagement of CD81 thereby blocking downstream SYK activation, which we have recently demonstrated to be important for effective HCV infection of target hepatocytes. Our findings highlight a novel functional class of anti-HCV agents that can inhibit HCV infection, most likely by disrupting vital viral-host signaling interactions at the level of virus entry. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS), the commonest malignancy of osteoarticular origin, is a very aggressive neoplasm. Divergent histologic differentiation is common in OS; hence triple diagnostic approach is essential in all cases. 20% cases are atypical owing to lack of concurrence among clinicoradiologic and pathologic features necessitating resampling. Recognition of specific anatomic and histologic variants is essential in view of better outcome. Traditional prognostic factors of OS do stratify patients for short term outcome, but often fail to predict their long term outcome. Considering the negligible improvement in the patient outcome during the last 20 years, search for novel prognostic factors is in progress like ezrin vascular endothelial growth factor, chemokine receptors, dysregulation of various micro ribonucleic acid are potentially promising. Their utility needs to be validated by long term followup studies before they are incorporated in routine clinical practice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-κB 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-κB in EGF-treated TSCC cells. Akt and NF-κB 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-κB 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. [ABSTRACT FROM AUTHOR]

Purpose: Ezrin is known to be involved in intercellular interactions, and a shift from membrane-bound to cytoplasmatic protein expression has been associated with malignant potential. This association has primarily been demonstrated in cell lines and, as yet, little is known about the distribution of ezrin in primary benign and malignant breast tissues. We have, therefore, set out to investigate ezrin protein expression in a series of primary breast lesions. Methods: Immunohistochemistry was used to detect ezrin expression in 465 samples of normal breast tissues, benign breast tumours, pre-invasive breast lesions, breast cancer tissues and metastatic lymph nodes, and the protein expression patterns observed were correlated with clinicopathological parameters. Results: Ezrin was detected in the cytoplasm of both benign and malignant breast tissues, but its expression was significantly higher in the malignant tissues (13 % vs 60 %, p < 0.0001; χ2 test). We also detected a statistically significant higher ezrin expression in pre-invasive lesions compared to benign lesions (15 % vs 44 %, p = 0.04; χ2 test). We did not find such a difference in ezrin expression between pre-invasive and invasive cancer samples, nor between invasive cancer samples and lymph node metastases. Within the group of invasive cancer samples, we found a significant correlation between ezrin expression and CK14 (rs:0.38, p < 0.007) and Her2 (rs:0.25, p < 0.002) expression. No such correlation was observed between ezrin expression and nodal status, grading, patient’s age, hormone receptor status, and Ki67 or p53 expression. Conclusion: Taken together, we found that cytoplasmatic ezrin expression increases from benign to malignant breast tumour development. We hypothesize that the tissue architectural alterations that are associated with aberrant ezrin expression may point at pathophysiological mechanisms that may be instrumental for the design of novel therapies. [ABSTRACT FROM AUTHOR]

L1, a neuronal cell adhesion receptor of the immunoglobulin-like protein family is expressed in invading colorectal cancer (CRC) cells as a target gene of Wnt/β-catenin signaling. Overexpression of L1 in CRC cells enhances cell motility and proliferation, and confers liver metastasis. We recently identified ezrin and the IκB-NF-κB pathway as essential for the biological properties conferred by L1 in CRC cells. Here, we studied the underlying molecular mechanisms and found that L1 enhances ezrin phosphorylation, via Rho-associated protein kinase (ROCK), and is required for L1-ezrin co-localization at the juxtamembrane domain and for enhancing cell motility. Global transcriptomes from L1-expressing CRC cells were compared with transcriptomes from the same cells expressing small hairpin RNA (shRNA) to ezrin. Among the genes whose expression was elevated by L1 and ezrin we identified insulin-like growth factor-binding protein 2 (IGFBP-2) and showed that its increased expression is mediated by an NF-κB-mediated transactivation of the IGFBP-2 gene promoter. Expression of a constitutively activated mutant ezrin (Ezrin567D) could also increase IGFBP-2 levels in CRC cells. Overexpression of IGFBP-2 in CRC cells lacking L1-enhanced cell proliferation (in the absence of serum), cell motility, tumorigenesis and induced liver metastasis, similar to L1 overexpression. Suppression of endogenous IGFBP-2 in L1-transfected cells inhibited these properties conferred by L1. We detected IGFBP-2 in a unique organization at the bottom of human colonic crypts in normal mucosa and at increased levels throughout human CRC tissue samples co-localizing with the phosphorylated p65 subunit of NF-κB. Finally, we found that IGFBP-2 and L1 can form a molecular complex suggesting that L1-mediated signaling by the L1-ezrin-NF-κB pathway, that induces IGFBP-2 expression, has an important role in CRC progression. [ABSTRACT FROM AUTHOR]

Ezrin is a member of the ezrin-radixin-moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell-matrix recognition, cell-cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling. [ABSTRACT FROM AUTHOR]

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer. [ABSTRACT FROM AUTHOR]

Various ependymoglial cells display varying degrees of process specialization, in particular processes contacting mesenchymal borders (pia, blood vessels, vitreous body), or those lining the ventricular surface. Within the neuropil, glial morphology, cellular contacts, and interaction partners are complex. It appears that glial processes contacting neurons, specific parts of neurons, or mesenchymal or ventricular borders are characterized by specialized membranes. We propose a concept of membrane domains in addition to the existing concept of ependymoglial polarity. Such membrane domains are equipped with certain membrane-bound proteins, enabling them to function in their specific environment. This review focuses on Müller cells and astrocytes and discusses exemplary the localization of established glial markers in membrane domains. We distinguish three functional glial membrane domains based on their typical molecular arrangement. The domain of the endfoot specifically displays the complex of dystrophin-associated proteins, aquaporin 4 and the potassium channel Kir4.1. We show that the domain of microvilli and the peripheral glial process in the Müller cell share the presence of ezrin, as do peripheral astrocyte processes. As a third domain, the Müller cell has peripheral glial processes related to a specific subtype of synapse. Although many details remain to be studied, the idea of glial membrane domains may permit new insights into glial function and pathology. [ABSTRACT FROM AUTHOR]

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P2 at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue. [ABSTRACT FROM AUTHOR]

Objectives: We have shown that ezrin expression correlates with ovarian epithelial cancer (OVCA) cell proliferation and metastatic behavior. In this study, we evaluated ezrin expression in transformed ovarian superficial epithelial cells (OSE) in ovarian clefts and in culture. Study Design: Immunohistochemistry and Western blotting for immunoreactive ezrin (ir-ezrin) in normal ovarian tissue, cultured OSE, and ovarian epithelial cancer cells. Results: While ir-ezrin was not demonstrable in normal cuboidal surface cells or interior ovarian organelles, cells lining the ovarian clefts strongly expressed ir-ezrin. Long-term culture of OSE increased ezrin expression and cytological abnormalities. Administration of estradiol and insulin at levels reported in inclusions dramatically induced OSE ir-ezrin expression to OVCA levels and membrane specializations; ruffling, pseudopodia and filopodia. Moreover epidermal growth factor (EGF) drastically increased ezrin translocation in OSE cells in a time-dependent manner. Conclusions: Ezrin expression by OSE increases during transformation.  Ezrin expression is responsive to estradiol and growth factors previously shown to be present in ovarian inclusions. These findings suggest that the microenvironment in ovarian inclusions and clefts contributes to the development of OVCA. Our findings elaborate on the mechanism of the ovarian origin of OVCA. [ABSTRACT FROM AUTHOR]

Purpose: Improved outcome after rectal cancer surgery requires identification of novel risk factors of tumour recurrence in order to personalise therapy, that is, enhanced selection of high-risk patients to additional radiochemotherapy or intensified follow-up. In several tumour types, including colorectal cancer, high expression of the membrane-cytoskeleton linker ezrin has been suggested to impair prognosis but has not yet reached clinical application. We evaluated the expression of ezrin in rectal cancer with a focus on the identification of a marker for local tumour recurrence. Methods: Immunohistochemical expression of ezrin was analysed in 104 primary rectal cancers from patients who developed local recurrences despite being treated with R0 major abdominal surgery. Time to local recurrence and distant metastasis as well as 5-year overall and cancer-specific survival were used as end points. Results: Ezrin expression was weak in 17% of the tumours, moderate in 62%, and intense in 21%. The time to local recurrence was significantly shorter ( p = 0.0004) for patients with tumours showing high ezrin expression. No correlation between ezrin expression and time to distant metastasis was identified. Survival data were similar between groups irrespective of ezrin expression in the primary tumours. Conclusions: Our findings suggest that increased expression of ezrin may represent a marker of aggressive biological behaviour in rectal cancer. Although further validation is needed, ezrin may represent a relevant marker for personalised treatment of rectal cancer with respect to risk of local recurrence after R0 surgery. [ABSTRACT FROM AUTHOR]

Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins. High ezrin expression is associated with lung metastasis and poor survival in cancer. We screened small molecule libraries for compounds that directly interact with ezrin protein using surface plasmon resonance to identify lead compounds. The secondary functional assays used for lead compound selection included ezrin phosphorylation as measured by immunoprecipitation and in vitro kinase assays, actin binding, chemotaxis, invasion into an endothelial cell monolayer, zebrafish and Xenopus embryonic development, mouse lung organ culture and an in vivo lung metastasis model. Two molecules, NSC305787 and NSC668394, that directly bind to ezrin with low micromolar affinity were selected based on inhibition of ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin-actin interaction and ezrin-mediated motility of osteosarcoma (OS) cells in culture. NSC305787 mimicked the ezrin morpholino phenotype, and NSC668394 caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of OS cells into mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. The small molecule inhibitors NSC305787 and NSC668394 demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis. [ABSTRACT FROM AUTHOR]

Uterine epithelial cells (UECs) undergo extensive morphological remodelling in preparation for an implanting blastocyst. This remodelling involves changes in the actin cytoskeleton and surface structures including microvilli. Ezrin and ezrin-radixin-moesin-binding protein-50-kDa (EBP50) link actin filaments to intra-membranous adhesion molecules and are important molecules in polarised epithelia. The current study is the first to describe the colocalisation and molecular association of ezrin and EBP50 in rat UECs by using immunofluorescence microscopy and immunoprecipitation techniques. These proteins have also been localised in relation to uterine epithelial cytoskeletal rearrangement during early pregnancy in the rat and to the effect of apical surface contact between opposing epithelial cells, blastocyst contact and contact with a silicon filament. Immunofluorescence microscopy has revealed that ezrin and EBP50 respond to contact between opposing epithelial cells and increase apically on day 6 of pregnancy. This apical distribution is also observed in UECs in contact with a silicon filament. Ezrin and EBP50 are however absent within the implantation chamber itself, seemingly mimicking the events that take place in leucocyte-endothelium binding. Thus, ezrin and EBP50 occur apically in UECs at the time of implantation in the rat and in response to a substitute blastocyst (filament) suggesting a role for these proteins in the cytoskeletal rearrangements that facilitate uterine receptivity and blastocyst-epithelial adhesion. Their loss within the implantation chamber possibly allows the subsequent invasion of the embryo. [ABSTRACT FROM AUTHOR]

Fibroblast adhesion can be modulated by proteins released by neuroendocrine cells and neurons, such as chromogranin A (CgA) and its N-terminal fragment vasostatin-1 (VS-1, CgA1–78). We have investigated the mechanisms of the interaction of VS-1 with fibroblasts and of its pro-adhesive activity and have found that the proadhesive activity of VS-1 relies on its interaction with the fibroblast membrane via a phospholipid-binding amphipathic α-helix located within residues 47–66, as well as on the interaction of the adjacent C-terminal region 67–78, which is structurally similar to ezrin–radixin–moesin-binding phosphoprotein 50 (a membrane-cytoskeleton adapter protein), with other cellular components critical for the regulation of cell cytoskeleton. [ABSTRACT FROM AUTHOR]

The forced overexpression of c-Myc in mouse prostate and in normal human prostate epithelial cells results in tumor transformation with an invasive phenotype. How c-Myc regulates cell invasion is poorly understood. In this study, we have investigated the interplay of c-Myc and androgens in the regulation of prostate cancer cell invasion. We found that c-Myc induces cell invasion and anchorage-independent growth by regulating ezrin protein expression in the presence of androgens. The activity of the ezrin promoter is controlled by androgens through c-Myc, which binds to a phylogenetically conserved E-Box located in the proximal promoter region. Besides, we also show that ezrin is an important regulator of c-Myc protein levels. These effects are achieved through androgen-induced changes in ezrin phosphorylation, which results in the regulation of downstream signals. These downstream signals involve the modulation of Akt and GSK-3β activity resulting in increased c-Myc protein synthesis and inhibition of its degradation. In summary, we have shown a key role for ezrin as a mediator of c-Myc-induced tumorigenesis in prostate cancer cells. [ABSTRACT FROM AUTHOR]

Ezrin links cortical actin filaments with the cell membrane, and has a critical role in many membrane-initiated events. Fas is directly associated with ezrin, but conflicting results have been reported for the involvement of ezrin in Fas-induced cell death. In this study we show that ezrin was associated with Fas in T cells before stimulation and was released shortly after Fas ligand (FasL) engagement. The knockdown of ezrin moderately increased Fas-triggered or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-triggered cell death in normal T lymphocytes and in H9 cells, but had no effect on death receptor-induced apoptosis in type II cells, such as Jurkat and CEM. Expression of a dominant-negative form of ezrin also led to an increased Fas-induced apoptosis in H9 cells. Ezrin deficiency did not affect the internalization of Fas after Fas ligation. Instead, an enhanced formation of death-inducing signaling complex (DISC) was observed in H9 cells with ezrin knockdown, leading to accelerated caspase-8 activation. Together, our results suggest that ezrin has a negative role in the recruitment of Fas into signaling complexes in type I T cells. Loss of ezrin likely removes the constraint imposed by ezrin and facilitates the assembly of death receptor complex in T cells. [ABSTRACT FROM AUTHOR]

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 β4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of β4 integrin. Suppression of β4 integrin expression by shRNA and disruption of β4 integrin function by transfection of dominant-negative β4 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 β4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between β4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The β4 integrin–ezrin interaction appears to be critical for maintenance of β4 integrin expression. These data begin to integrate ezrin and β4 integrin expression into a model of action for the mechanism of osteosarcoma metastases. [ABSTRACT FROM AUTHOR]

Tumor metastasis is a complex phenomenon that is the culmination of effects of numerous cellular factors. We have shown that the Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is capable of inducing a wide range of such factors in cell culture, expression of which is also elevated in the LMP1-expressing tumor, nasopharyngeal carcinoma (NPC), a highly invasive neoplasm. Recently, the membrane crosslinker protein, ezrin, has been implicated in tumor cell metastasis and malignant progression. In this study, we evaluated the possible role of LMP1 and ezrin in the pathophysiology of NPC. We show that C-terminal phosphorylation of ezrin is increased by the expression of LMP1 in nasopharyngeal (NP) cells through a protein kinase C (PKC) pathway. LMP1 enhances the organization of a ternary complex of CD44, ezrin and F-actin, which is a prerequisite for ezrin phosphorylation. In NPC tissues, the expression of phosphoezrin and LMP1 is directly correlated. Silencing of endogenously expressed ezrin suppresses LMP1-induced cell motility and invasiveness. Moreover, the inhibition of ezrin phosphorylation by PKC inhibitor suppresses migration and invasion of NP cells. These data show that the phosphorylation of ezrin and its recruitment to the cell membrane linked to F-actin and CD44 is a process required for LMP1-stimulated cell motility and invasion of NP cells.Oncogene (2009) 28, 1725–1735; doi:10.1038/onc.2009.20; published online 23 February 2009 [ABSTRACT FROM AUTHOR]

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 PKCα, PKCι and PKCγ. 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.Oncogene (2009) 28, 792–802; doi:10.1038/onc.2008.437; published online 8 December 2008 [ABSTRACT FROM AUTHOR]

This review explores possible mechanisms by which the neurofibromatosis type-2 tumour suppressor Merlin regulates contact-dependent inhibition of proliferation. Starting from an evolutionary perspective, the concurrent emergence of intercellular contacts and proliferation control in multicellular organisms is first considered. Following a brief survey of the molecular and subcellular milieus in which merlin performs its function, the importance of different cellular and biological contexts in defining the function of merlin is discussed. Finally, an integrated model for merlin and the Ezrin, Radixin, and Moesin (ERM) proteins functioning in the regulation of cellular interfaces is proposed. [ABSTRACT FROM AUTHOR]

The change of cell mobility is one of the preconditions of tumor metastasis. Cell skeleton alteration and rearrangement of F-actin was closely related to cell mobility. Ezrin is a membrane-cytoskeleton organizer that can mediate the rearrangement and the function of F-actin. In this paper, we investigated the effect of ezrin on hepatocellullar carcinoma cell growth and invasiveness. Hepatocellular carcinoma cell lines such as MHCC-1, MHCC97-H, SF7721, SMMC7721, Hep3B, and HepG2 were chosen in this study. We first examined the expression and the distribution of ezrin and F-actin in these cell lines using immunofluorescence, RT–PCR, and the western blot. Next we used small interfering RNA (siRNA) to down-regulate ezrin expression in MHCC-1, MHCC97-H, SF7721, and HepG2 to investigate the role of ezrin in tumor cell growth and invasiveness. Our preliminary results showed that the expression of ezrin and γ-actin in MHCC-1, MHCC97-H, and SF7721 with higher metastatic potential were obviously up-regulated than those in SMMC7721, Hep3B, and HepG2 with lower potential. No different expression of β-actin was found in the above tumor cell lines. The outcome of RNAi indicated that decreasing ezrin expression can notably inhibit the proliferation of the four hepatocellular carcinoma cell lines ( p < 0.01, n = 10). The proportion of cells in G2-M phase also decreased after RNAi. The number of pseudopods decreased as well after RNAi treatment ( p < 0.01, n = 5). The mobility and invasiveness of cancer cells decreased with decreasing ezrin expression tested by transwell assay ( p < 0.01, n = 8). Ezrin plays an important role in the process of hepatocellular carcinoma cell proliferation, migration, and invasiveness. [ABSTRACT FROM AUTHOR]

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4+T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4+T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.Cell Death and Differentiation (2004) 11, 574-582. doi:10.1038/sj.cdd.4401374 Published online 23 January 2004 [ABSTRACT FROM AUTHOR]

The skin is an interesting organ for human gene therapy due to accessibility, immunologic potential and synthesis capabilities. In this study, we attempted to visualize and measure the uptake of naked FITC-labeled plasmid by FACS analysis detecting up to 15% internalization in a dose- and time-dependent manner. Cycloheximide treatment inhibited the uptake by >90%, suggesting a protein-mediated uptake. The inhibition of different internalization pathways demonstrated that blocking macropinocytosis (by amiloride and N,N-dimethylamylorid) reduced DNA uptake by >85%, while the inhibition of clathrin-coated pits (by chlorpromazine) and caveolae (by nystatin/filipin III) did not limit the uptake. Colocalization studies using confocal laser microscopy revealed a time-dependent accumulation of plasmid DNA in endosomes and lysosomes. When a green fluorescent protein (GFP) expression vector was used, specific GFP-RNA became detectable by reverse transcriptase-PCR, whereas measurable amounts of protein could not be identified in FACS experiments. To detect the potential DNA receptors on the keratinocyte surface, membrane proteins were extracted and subjected to South-Western blotting using digoxigenin-labeled calf thymus and ?-phage DNA. Two DNA-binding proteins, ezrin and moesin, known as plasma membrane-actin linkers, were identified by one- and two-dimensional-South-Western blots and matrix-assisted laser desorption and ionization-mass spectrometry. Ezrin and moesin are functionally associated with a number of transmembrane receptors such as the EGF, CD44 or ICAM-1 receptor. Taken together, naked plasmid DNA seems to enter human keratinocytes through different pathways, mainly by macropinocytosis. Two DNA-binding proteins were identified that seemed to be involved in binding/trafficking of internalized DNA.Gene Therapy (2004) 11, 765-774. doi:10.1038/sj.gt.3302221 Published online 15 January 2004 [ABSTRACT FROM AUTHOR]

The article focuses on the study of functional organization of cell cortex by ezrin, radixin and moesin (ERM) proteins with tumor suppressor neurofibromatosis type 2 (NF2 or merlin). It comprised of localization of ezrin and NF2 in Matrigel-embedded intestinal epithelial cells. It was observed that ezrin did not localize to the cap structure whereas NF2 did.