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.     

ERM stable knockdown by siRNA reduced in vitro migration and invasion of human SGC-7901 cells

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

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.     

埃兹蛋白的表达与非小细胞肺癌转移的关系研究

目的:探究埃兹蛋白(Ezrin)的表达与非小细胞肺癌转移的关系。方法:通过免疫组化检测Ezrin在有无转移的非小细胞肺癌组织中的表达差异,通过细胞免疫组化、western-blot、RT-PCR检测Ezrin在不同转移潜能肺癌细胞系中的表达差异,通过transwell考察Ezrin对不同转移潜能癌细胞侵袭和迁移能力的影响。结果:Ezrin蛋白在有转移的非小细胞肺癌组织中的表达水平明显高于无转移的肺癌组织,在高转移潜能肺癌细胞系中的表达高于低转移潜能细胞系,受抑制时会削弱高转移潜能肺癌细胞的迁移和侵袭能力,过表达时会增强低转移潜能肺癌细胞的侵袭和迁移能力。结论:Ezrin可能在非小细胞肺癌及其转移中发挥重要作用。     

MicroRNA-100 regulates IGF1-receptor expression in metastatic pancreatic cancer cells

Abstract

Patients with pancreatic adenocarcinoma have the lowest 5 year survival rate and yearly rates of incidence are nearly equal to the mortality rates. Long term cure rates by standard therapies are disappointing owing to disseminated disease at diagnosis and chemotherapeutic resistance. New therapeutic targets are necessary to decrease the progression of pancreatic cancer and the ability to identify targets specific to metastasis would improve patient care. We evaluated the levels of microRNA of metastatic and non-metastatic cell lines. The expression levels of microRNAs and mRNAs were determined using microarray analysis to examine and compare five pancreatic cancer cell lines, two that can metastasize in vivo (S2VP10 and S2CP9) and three that do not metastasize (MiaPaCa2, Panc-1 and ASPC-1). MicroRNA analysis indicated an increase in miR-100 and a decrease in miR-138 expression in metastatic cancer cells. Microarray analysis of different expressions of mRNAs in metastatic and non-metastatic pancreatic cell lines also indicated significantly increased insulin growth factor-1 receptor (IGF1-R) expression in metastatic pancreatic cancer cell lines compared to non-metastatic pancreatic cancer cell lines. To confirm microarray analysis results, western blot and immunocytochemistry were performed. Western blot revealed that IGF1-R expression exhibited in metastatic cancer cell lines a seven-fold increase compared to non-metastatic cell lines. In addition, downstream expressions of the proteins, GRB2 and phosphorylated PI3K, also were increased in aggressive cancer cell lines. Immunocytochemistry confirmed the linkage of IGF1-R to miR-100, because cells transfected with miR-100 inhibitor showed a decrease in IGF1-R. Cells transfected with a miR-138 mimic, however, did not affect IGF1-R expression.     

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells.     

13q14断裂重排与非小细胞肺癌转移潜能关系的研究

肿瘤转移的细胞经常存在染色体数目异常和结构畸变,在多种有转移潜能的肿瘤细胞中都涉及到13q14的异常。以往研究表明在同一组织来源但转移潜能不同的肺腺癌细胞系AGZY83-a和Anip973中存在13q14的断裂重排。采用mRNA差异展示技术（mRNA DD）分析这一对细胞系得到的差异表达基因BRI基因位于13q14。为了进一步分析肺癌细胞的转移潜能与13q14断裂重排间的关系,采用13q涂染探针对具有不同转移潜能的非小细胞肺癌细胞系PAa、SPC-1-A和95D中期分裂相进行G显带后的荧光原位杂交分析。结果发现在3个肺癌细胞系中有多种13号染色体长臂的结构异常,其中此3个细胞系均涉及13q32-33的频发断裂。但是低转移肺癌细胞系PAa、SPC-1-A均未涉及13q14的断裂,而高转移肺癌细胞系95D的两种细胞克隆均可见13q14的断裂。提示13q14断裂点与肺癌细胞的转移能力有一定的相关性,两者之间的遗传学意义需要进一步研究探索。     

Ezrin Ser66 phosphorylation regulates invasion and metastasis of esophageal squamous cell carcinoma cells by mediating filopodia formation

BackgroundEzrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear.MethodsDistribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis.ResultsEzrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo.ConclusionEzrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.     

ICAM-2 expression mediates a membrane-actin link, confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma

The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/alpha-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/alpha-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.     

Quantitative Proteomic Profiling Identifies DPYSL3 as Pancreatic Ductal Adenocarcinoma-Associated Molecule That Regulates Cell Adhesion and Migration by Stabilization of Focal Adhesion Complex

Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.     

埃兹蛋白:生物学特征及其在肿瘤转移中的作用

埃兹蛋白(ezrin)是埃兹蛋白、根蛋白和膜突蛋白(ezrin-radixin-moesin,ERM)家族成员之一,主要参与上皮细胞中细胞骨架与胞膜之间的连接,具有维持细胞形态和运动、连接黏附分子及调节信号转导等功能。近年来的研究发现,埃兹蛋白在肿瘤细胞中的表达异常,提示其在肿瘤的浸润、转移机制中发挥重要作用。     

Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fourth leading cause for cancer-related mortality. Stem cells have been implicated in pancreatic tumor growth, but the specific role of these cancer stem cells in tumor biology, including metastasis, is still uncertain. We found that human pancreatic cancer tissue contains cancer stem cells defined by CD133 expression that are exclusively tumorigenic and highly resistant to standard chemotherapy. In the invasive front of pancreatic tumors, a distinct subpopulation of CD133(+) CXCR4(+) cancer stem cells was identified that determines the metastatic phenotype of the individual tumor. Depletion of the cancer stem cell pool for these migrating cancer stem cells virtually abrogated the metastatic phenotype of pancreatic tumors without affecting their tumorigenic potential. In conclusion, we demonstrate that a subpopulation of migrating CD133(+) CXCR4(+) cancer stem cells is essential for tumor metastasis. Strategies aimed at modulating the SDF-1/CXCR4 axis may have important clinical applications to inhibit metastasis of cancer stem cells.     

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.     

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.     

Characterization of the Ca2+ -regulated ezrin-S100P interaction and its role in tumor cell migration

Ezrin is a multidomain protein providing regulated membrane-cytoskeleton contacts that play a role in cell differentiation, adhesion, and migration. Within the cytosol of resting cells ezrin resides in an autoinhibited conformation in which the N- and C-terminal ezrin/radixin/moesin (ERM) association domains (ERMADs) interact with one another. Activation of the ezrin membrane-cytoskeleton linker function requires an opening of this interdomain association that can result from phosphatidylinositol 4,5-bisphosphate binding to the N-ERMAD and threonine 567 phosphorylation in the C-ERMAD. We have shown that ezrin can also be activated by Ca(2+)-dependent binding of the EF-hand protein S100P. We now provide a quantitative analysis of this interaction and map the respective binding sites to the F2 lobe in the ezrin N-ERMAD and a stretch of hydrophobic residues in the C-terminal extension of S100P. Phospholipid binding assays reveal that S100P and phosphatidylinositol 4,5-bisphosphate compete to some extent for at least partially overlapping binding sites in N-ERMAD. Using interaction-competent as well as interaction-incompetent S100P derivatives and permanently active ezrin mutants, we also show that the protein interaction and a resulting activation of ezrin promote the transendothelial migration of tumor cells. Thus, a prometastatic role of ezrin and S100P that had been proposed based on their overexpression in highly metastatic cancers is probably due to a direct interaction between the two proteins and the S100P-mediated activation of ezrin.     

Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.     

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.     

Molecular characteristics and chromatin texture features in acute promyelocytic leukemia

Background

Ezrin is a cytoskeletal protein that is involved in tumor growth and invasion. It has been suggested that Ezrin expression plays an important role in tumor metastasis. This study is aimed to investigate the clinicopathological significance of Ezrin overexpression in gastric adenocarcinomas.

Methods

Ezrin protein expression was examined by immunohistochemistry in 26 normal gastric mucosa, 32 dysplasia, and 277 gastric adenocarcinomas. The relationship between Ezrin expression and the clinicopathological features of gastric cancers was analyzed. In addition, a gastric cancer cell line, MKN-1, was also used for immunofluorescence staining to evaluate the distribution of Ezrin protein.

Results

Ezrin protein located in the cytoplasm and/or membrane in the migrating gastric cancer cells, and it mainly concentrated at the protrusion site; however, only cytoplasmic distribution was observed in the non-migrating cancer cells by immunofluorescence staining. The positive rate of Ezrin protein expression was significantly higher in gastric adenocarcinoma and dysplasia compared with that in the normal gastric mucosa. Moreover, expression frequency of Ezrin protein increased significantly in lymph node metastasis and late clinical stages. Consistently, strong expression of Ezrin was significantly correlated with poor prognosis of gastric cancer.

Conclusion

The detection of Ezrin expression can be used as the marker for early diagnosis and prognosis of gastric adenocarcinoma.

Virtual Slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2303598677653946