The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis

Metastatic cancers, once established, are the primary cause of mortality associated with cancer. Previously, we used a genomic approach to identify metastasis-associated genes in cancer. From this genomic data, we selected ezrin for further study based on its role in physically and functionally connecting the actin cytoskeleton to the cell membrane. In a mouse model of osteosarcoma, a highly metastatic pediatric cancer, we found ezrin to be necessary for metastasis. By imaging metastatic cells in the lungs of mice, we showed that ezrin expression provided an early survival advantage for cancer cells that reached the lung. AKT and MAPK phosphorylation and activity were reduced when ezrin protein was suppressed. Ezrin-mediated early metastatic survival was partially dependent on activation of MAPK, but not AKT. To define the relevance of ezrin in the biology of metastasis, beyond the founding mouse model, we examined ezrin expression in dogs that naturally developed osteosarcoma. High ezrin expression in dog tumors was associated with early development of metastases. Consistent with this data, we found a significant association between high ezrin expression and poor outcome in pediatric osteosarcoma patients.     

High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential.

Ezrin is a membrane cytoskeleton crosslinker protein that is a member of the ERM (ezrin/radixin/moesin) family. Ezrin binds adhesion molecules such as CD43, CD44, ICAM-1, and ICAM-2, which are implicated in cell migration and metastasis. Ezrin is expressed by many tumor cell lines; however, little is known about the function of ezrin in tumorigenesis and metastasis. Here, we investigated expression of ezrin in pancreatic adenocarcinoma cell lines of different metastatic potential. Among 16 pancreatic adenocarcinoma cell lines, several cell lines showed strong expression of ezrin. Two cell lines with high metastatic potential, S2-CP9 and S2-VP10, showed very high levels of ezrin mRNA and protein, whereas other sublines showed lower levels. There was no relationship between the expression levels of ezrin and the differentiation grades of the cell lines. These results suggest that there is a relationship between high expression of ezrin and metastatic potential of pancreatic carcinomas.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

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.     

Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion

Up-regulation of the cytoskeleton linker protein ezrin frequently occurs in aggressive cancer types and is closely linked with metastatic progression. However, the underlying molecular mechanisms detailing how ezrin is involved in the invasive and metastatic phenotype remain unclear. Here we report a novel function of ezrin in regulating focal adhesion (FA) and invadopodia dynamics, two key processes required for efficient invasion to occur. We show that depletion of ezrin expression in invasive breast cancer cells impairs both FA and invadopodia turnover. We also demonstrate that ezrin-depleted cells display reduced calpain-mediated cleavage of the FA and invadopodia-associated proteins talin, focal adhesion kinase (FAK), and cortactin and reduced calpain-1–specific membrane localization, suggesting a requirement for ezrin in maintaining proper localization and activity of calpain-1. Furthermore, we show that ezrin is required for cell directionality, early lung seeding, and distant organ colonization but not primary tumor growth. Collectively our results unveil a novel mechanism by which ezrin regulates breast cancer cell invasion and metastasis.     

PTEN Loss Increases PD-L1 Protein Expression and Affects the Correlation between PD-L1 Expression and Clinical Parameters in Colorectal Cancer

Background

Programmed death ligand-1 (PD-L1) has been identified as a factor associated with poor prognosis in a range of cancers, and was reported to be mainly induced by PTEN loss in gliomas. However, the clinical effect of PD-L1 and its regulation by PTEN has not yet been determined in colorectal cancer (CRC). In the present study, we verified the regulation of PTEN on PD-L1 and further determined the effect of PTEN on the correlation between PD-L1 expression and clinical parameters in CRC.

Methods/Results

RNA interference approach was used to down-regulate PTEN expression in SW480, SW620 and HCT116 cells. It was showed that PD-L1 protein, but not mRNA, was significantly increased in cells transfected with siRNA PTEN compared with the negative control. Moreover, the capacity of PTEN to regulate PD-L1 expression was not obviously affected by IFN-γ, the main inducer of PD-L1. Tissue microarray immunohistochemistry was used to detect PD-L1 and PTEN in 404 CRC patient samples. Overexpression of PD-L1 was significantly correlated with distant metastasis (P<0.001), TNM stage (P<0.01), metastatic progression (P<0.01) and PTEN expression (P<0.001). Univariate analysis revealed that patients with high PD-L1 expression had a poor overall survival (P<0.001). However, multivariate analysis did not support PD-L1 as an independent prognostic factor (P = 0.548). Univariate (P<0.001) and multivariate survival (P<0.001) analysis of 310 located CRC patients revealed that high level of PD-L1 expression was associated with increased risks of metastatic progression. Furthermore, the clinical effect of PD-L1 on CRC was not statistically significant in a subset of 39 patients with no PTEN expression (distant metastasis: P = 0.102; TNM stage: P = 0.634, overall survival: P = 0.482).

Conclusions

PD-L1 can be used to identify CRC patients with high risk of metastasis and poor prognosis. This clinical manifestation may be partly associated with PTEN expression.     

Fascin-1, Ezrin and Paxillin Contribute to the Malignant Progression and Are Predictors of Clinical Prognosis in Laryngeal Squamous Cell Carcinoma

Aims

Fascin-1, ezrin and paxillin, cytoskeleton-associated proteins, have been implicated in several human cancers, but their role in laryngeal squamous cell carcinoma (LSCC) is unknown. We investigated the association of their expression and clinicopathologic factors and their prognostic value in LSCC.

Materials and Methods

Quantitative RT-PCR and western blot analyses were used to examine mRNA and protein levels in 10 fresh LSCC specimens and 10 corresponding adjacent normal margin (ANM) tissues from patients undergoing surgery in 2012. We used immunohistochemistry to retrospectively study 216 paraffin blocks of LSCC samples from patients (193 men) who had undergone surgery between 2000 and 2006 and had not received special treatment before the diagnosis. Univariate analysis of patient survival involved the Kaplan–Meier method. Multivariate analyses involved the Cox proportional hazards model.

Results

The relative mRNA and protein levels of fascin-1, ezrin and paxillin were significantly greater in LSCC than ANM tissue (P<0.05). The high expression of fascin-1, ezrin or paxillin was positively correlated with poor tumor differentiation, cervical lymph node metastasis (N+), and advanced clinical stage (III+IV) (P<0.05) but not sex or metastasis. In addition, a high expression of fascin-1 (P = 0.007) or ezrin (P = 0.047) was associated with advanced tumor stage (T3+T4). The expression of fascin-1 was higher in smokers than non-smokers (P = 0.019). A high expression of fascin-1, ezrin or paxillin was associated with poor prognosis.

Conclusions

Fascin-1, ezrin and paxillin may be prognostic of poor outcome with LSCC after surgery. Our study may lead to establishing new molecular therapeutic targets and/or prognostic biomarkers in LSCC.     

Epigenetic Drugs Can Stimulate Metastasis through Enhanced Expression of the Pro-Metastatic Ezrin Gene

Ezrin has been reported to be upregulated in many tumors and to participate in metastatic progression. No study has addressed epigenetic modification in the regulation of Ezrin gene expression, the importance of which is unknown. Here, we report that highly metastatic rhabdomyosarcoma (RMS) cells with high levels of Ezrin have elevated acetyl-H3-K9 and tri-methyl-H3-K4 as well as reduced DNA methylation at the Ezrin gene promoter. Conversely, poorly metastatic RMS cells with low levels of Ezrin have reduced acetyl-H3-K9 and elevated methylation. Thus epigenetic covalent modifications to histones within nucleosomes of the Ezrin gene promoter are linked to Ezrin expression, which in fact can be regulated by epigenetic mechanisms. Notably, treatment with histone deacetylase (HDAC) inhibitors or DNA demethylating agents could restore Ezrin expression and stimulate the metastatic potential of poorly metastatic RMS cells characterized by low Ezrin levels. However, the ability of epigenetic drugs to stimulate metastasis in RMS cells was inhibited by expression of an Ezrin-specific shRNA. Our data demonstrate the potential risk associated with clinical application of broadly acting covalent epigenetic modifiers, and highlight the value of combination therapies that include agents specifically targeting potent pro-metastatic genes.     

Proteome analysis of human androgen-independent prostate cancer cell lines: variable metastatic potentials correlated with vimentin expression

To better understand the molecular mechanisms of prostate cancer (PCA) dissemination and to develop new anti-metastasis therapies, key regulatory molecules involved in PCA metastasis were identified in two human androgen-independent PCA cell lines, highly metastatic 1E8-H and lowly metastatic 2B4-L cells. Through 2-DE and MS analyses, 12 proteins with different expression levels in the two cell lines were identified. The following proteins were found to be significantly up-regulated in 1E8-H cells compared with 2B4-L cells: gp96 precursor, calreticulin precursor, vimentin (VIM), Hsp90alpha, peroxiredoxin 2, HNRPH1, ezrin, T-complex protein 1, alpha subunit, and hypothetical protein mln2339. In contrast, heart L-lactate dehydrogenase H chain, annexin I, and protein disulfide isomerase were notably down-regulated in 1E8-H cells compared with 2B4-L cells. To our knowledge, this study is the first to demonstrate that up-regulation of VIM expression positively correlates with the invasion and metastasis of androgen-independent PCA.     

Ezrin expression and cell survival regulation in colorectal cancer

Colorectal cancer (CRC) is the second largest cause of cancer deaths in the United States. A key barrier that prevents better outcomes for this type of cancer as well as other solid tumors is the lack of effective therapies against the metastatic disease. Thus there is an urgent need to fill this gap in cancer therapy. We utilized a 2D-DIGE proteomics approach to identify and characterize proteins that are differentially regulated between primary colon tumor and liver metastatic deposits of the IGF1R-dependent GEO human CRC xenograft, orthotopically implanted in athymic nude mice that may serve as potential therapeutic targets against CRC metastasis. We observed increased expression of ezrin in liver metastasis in comparison to the primary colonic tumor. Increased ezrin expression was further confirmed by western blot and microarray analyses. Ezrin, a cytoskeletal protein belonging to Ezrin–Radixin–Moesin (ERM) family plays important roles in cell motility, invasion and metastasis. However, its exact function in colorectal cancer is not well characterized. Establishment of advanced GEO cell lines with enhanced liver-metastasizing ability showed a significant increase in ezrin expression in liver metastasis. Increased phosphorylation of ezrin at the T567 site (termed here as p-ezrin T567) was observed in liver metastasis. IHC studies of human CRC patient specimens showed an increased expression of p-ezrin T567 in liver metastasis compared to the primary tumors of the same patient. Ezrin modulation by siRNA, inhibitors and T567A/D point mutations significantly downregulated inhibitors of apoptosis (IAP) proteins XIAP and survivin that have been linked to increased aberrant cell survival and metastasis and increased cell death. Inhibition of the IGF1R signaling pathway by humanized recombinant IGF1R monoclonal antibody MK-0646 in athymic mouse subcutaneous xenografts resulted in inhibition of p-ezrin T567 indicating ezrin signaling is downstream of the IGF1R signaling pathway. We identified increased expression of p-ezrin T567 in CRC liver metastasis in both orthotopically implanted GEO tumors as well as human patient specimens. We report for the first time that p-ezrin T567 is downstream of the IGF1R signaling and demonstrate that ezrin regulates cell survival through survivin/XIAP modulation.     

肌源性调节蛋白MyoDl和肌球蛋白myosin在横纹肌肉瘤中的表达:免疫组织化学研究

应用免疫组织化学ＳＰ方法,检测了肌源性调节蛋白ＭｙｏＤｌ和肌球蛋白ｍ ｙｏｓｉｎ 在３８例横纹肌肉瘤（ＲＭＳ） 中的表达。结果显示ＭｙｏＤｌ阳性表达主要定位于ＲＭＳ瘤细胞的胞核中; ｍ ｙｏｓｉｎ 的阳性表达定位于ＲＭＳ瘤细胞的胞浆中, 二者的表达阳性率分别为６５．８％ 和５５．３％ 。在ＲＭＳ不同病理分型中ＭｙｏＤｌ和ｍ ｙｏｓｉｎ 的阳性表达均无显著性差异。但ＲＭＳ分化程度低,ＭｙｏＤｌ阳性表达增强,ｍ ｙｏｓｉｎ 阳性表达下降。Ｍｙｏ－Ｄｌ在Ⅲ级（低分化） 中的表达阳性率显著高于Ⅰ级（高分化）、Ⅱ级（中等分化） 中的表达阳性率（Ｐ＜ ０．０５,Ｐ＜ ０．０５）。ｍ ｙｏｓｉｎ 在Ⅰ级中的阳性率显著高Ⅲ级中的表达阳性率（Ｐ＜ ０．０５）。本文认为, ＭｙｏＤｌ表达增高、ｍ ｙｏｓｉｎ 表达下降,不仅是ＲＭＳ生物学行为的重要特征,也为改进低分化ＲＭＳ的病理诊断和ＲＭＳ早期诊断提供了有益的思路。     

Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain

Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NH2-terminal domain (ezrin 1-233) and that of a COOH-terminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Sf9 cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 microns in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlstrom, and A. Vaheri. 1994. J. Cell Biol. 126:1445- 1453), suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NH2- terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.     

Roles of the Cyclooxygenase 2 Matrix Metalloproteinase 1 Pathway in Brain Metastasis of Breast Cancer

Brain is one of the major sites of metastasis in breast cancer; however, the pathological mechanism of brain metastasis is poorly understood. One of the critical rate-limiting steps of brain metastasis is the breaching of blood-brain barrier, which acts as a selective interface between the circulation and the central nervous system, and this process is considered to involve tumor-secreted proteinases. We analyzed clinical significance of 21 matrix metalloproteinases on brain metastasis-free survival of breast cancer followed by verification in brain metastatic cell lines and found that only matrix metalloproteinase 1 (MMP1) is significantly correlated with brain metastasis. We have shown that MMP1 is highly expressed in brain metastatic cells and is capable of degrading Claudin and Occludin but not Zo-1, which are key components of blood-brain barrier. Knockdown of MMP1 in brain metastatic cells significantly suppressed their ability of brain metastasis in vivo, whereas ectopic expression of MMP1 significantly increased the brain metastatic ability of the cells that are not brain metastatic. We also found that COX2 was highly up-regulated in brain metastatic cells and that COX2-induced prostaglandins were directly able to promote the expression of MMP1 followed by augmenting brain metastasis. Furthermore, we found that COX2 and prostaglandin were able to activate astrocytes to release chemokine (C-C motif) ligand 7 (CCL7), which in turn promoted self-renewal of tumor-initiating cells in the brain and that knockdown of COX2 significantly reduced the brain metastatic ability of tumor cells. Our results suggest the COX2-MMP1/CCL7 axis as a novel therapeutic target for brain metastasis.     

Src-dependent ezrin phosphorylation in adhesion-mediated signaling

In addition to providing a regulated linkage between the membrane and the actin cytoskeleton, ezrin participates in signal transduction pathways. Here we describe that expression of the ezrin Y145F mutant delays epithelial cell spreading on fibronectin by inhibiting events leading to FAK activation. The defect in spreading was rescued by the overexpression of catalytically functional Src. We demonstrate that ezrin Y145 is phosphorylated in A431 cells stimulated with epidermal growth factor (EGF) and in v-Src-transformed cells. Moreover in cells devoid of Src, SYF-/- fibroblasts, ezrin Y145 phosphorylation could only be detected upon the introduction of an active form of Src. The phosphorylation of ezrin at Y145 required prior binding of the Src SH2 domain to ezrin. Our results further show that Src activity influences its binding to ezrin and a positive feedback mechanism for Src-mediated Y145 phosphorylation is implied. Interestingly, cells expressing ezrin Y145F did not proliferate when cultured in a 3D collagen gel. Collectively, our results demonstrate a key signaling input of Src-dependent ezrin phosphorylation in adhesion-mediated events in epithelial cells.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Ezrin controls the macromolecular complexes formed between an adapter protein Na+/H+ exchanger regulatory factor and the cystic fibrosis transmembrane conductance regulator

Na(+)/H(+) exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)(2).NHERF.ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR.NHERF complex. Immunoprecipitation and immunoblotting confirm the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton.     

Matriptase-2 inhibits HECV motility and tubule formation in vitro and tumour angiogenesis in vivo

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.     

Expression and Functional Significance of Ezrin in Human Brain Astrocytoma

Ezrin is overexpressed in a variety of neoplastic cells and is involved in the later stages of tumor progression and metastasis. The present study investigated the expression and functional significance of ezrin in human brain astrocytoma. Ezrin expression was examined in specimens from healthy human brains (10 autopsies) or human astrocytoma (107 cases) by immunohistochemistry. All healthy specimens were negative for ezrin expression, while this expression was positive in a great majority of human astrocytoma tissues (96/107; 89.7 %; p < 0.05 vs. healthy). Ezrin expression was positively correlated with tumor grade (r = 0.551, p < 0.01). Analysis of clinicopathologic data revealed that the post-operation disease-free survival times were significantly (p < 0.001) different between those with a strong positive ezrin expression and those with a weak or negative expression. Specifically, median DFS in patients with a strongly positive ezrin expression was 13 months (range 2–46 months), while it was significantly (p < 0.001) longer in patients with weakly positive or negative expression (median of 28 months, range 6–56 months). In conclusion, there is a strong association between ezrin expression and increased malignancy in astrocytoma. Thus, enhanced ezrin expression may play an important role in the development of astrocytoma. Our results further indicate that ezrin may be useful for grading of astrocytoma and as a molecular marker for the prognosis.