Ezrin-radixin-moesin-binding Phosphoprotein 50 Research Articles

Ezrin-Radixin-Moesin Binding Phosphoprotein 50 (EBP50) Suppresses the Metastasis of Breast Cancer and HeLa Cells by Inhibiting Matrix Metalloproteinase-2 Activity.

Expression of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) is correlated with human breast and cervical cancer development, but its effects on the metastasis of breast and cervical cancer and the underlying mechanism are not fully understood. In this study, EBP50 was overexpressed in MDA-MB-231 breast cancer and HeLa cervical cancer cells; moreover, EBP50 was knocked-down in MCF-7 breast cancer cells and HeLa cells. Metastasis-related ability and matrix metalloproteinase-2 (MMP-2) activity of these cells were investigated. Cell adhesion, wound-healing and invasion were significantly suppressed in EBP50-overexpressing cells. Contrarily, EBP50-knockdown promoted cell adhesion, wound healing and invasion. EBP50 overexpression inhibited MMP-2 activity, and the knockdown of EBP50 promoted the activity of MMP-2, suggesting that EBP50 inhibited cell metastasis via suppression of MMP-2 activity. Our work reveals the anti-metastatic effect and a new mechanism of EBP50 action in breast and cervical cancer cells.

Reduced EBP50 expression levels are correlated with unfavorable clinicopathological features of extrahepatic bile duct carcinoma and promote the proliferation and migration of QBC939 cells.

The present study aimed to clarify the association between ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) expression level and the tumor phenotype and clinicopathological features of extrahepatic bile duct carcinoma. Tissue samples from patients with extrahepatic bile duct carcinoma (54 cases) and patients with normal bile duct epithelia from gallbladder of cholecystitis (20 cases) were collected, and immunohistochemical staining was used to detect the expression levels of EBP50 in these tissues. In addition, small interfering (si)RNA-EBP50 was used to knock down the expression of EBP50 in the QBC939 human cholangiocarcinoma (CC) cell line. The effect of EBP50 expression on QBC939 cell proliferation and migration was analyzed using the Cell Counting kit-8 and wound healing assays, respectively. EBP50 expression was significantly downregulated in CC tissue samples (P<0.01), with low EBP50 expression levels positively correlated with a high pathological stage and a poor differentiation degree (P<0.01 and P<0.001, respectively). EBP50 expression in QBC939 cells was knocked down by ≤80% using siRNA-EBP50, and EBP50 knockdown significantly promoted QBC939 cell proliferation, as compared with the vector control cells (P=0.04). EBP50 knockdown also significantly enhanced the wound healing ability of QBC939 cells (P=0.02). These results demonstrated that EBP50 expression levels are significantly correlated with a malignant phenotype in patients with CC, and decreased expression levels of EBP50 may promote CC cell proliferation and migration. These findings provide insight into novel potential diagnostic and therapeutic approaches for patients with CC.

EBP50 interacts with EGFR and regulates EGFR signaling to affect the prognosis of cervical cancer patients.

Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) has a role in the occurrence and progression of multiple types of tumors. However, its role in cervical cancer(CC) remain unknown. EBP50 was reported to interact with epidermal growth factor receptor (EGFR) and regulate EGFR signaling in CC HeLa cells. In this study, the effect of EBP50 expression on CC cell proliferation and prognosis of CC patients by regulating EGFR signaling was investigated. We found that EBP50 expression level was significantly downregulated in CC tissues. EBP50 expression negatively correlated with CC cell proliferation, cell cycle and the activation of EGFR-mediated ERK signaling. EBP50 knockdown abolished its inhibition on EGF-induced ERK activation, suggesting EBP50 regulated EGFR signaling. In order to further explore EBP50 regulated EGFR signaling via interaction, we constructed EBP50_DD mutant which disrupted its interaction with EGFR. EBP50_DD overexpression attenuated the inhibition of EBP50_WT on EGFR-mediated ERK signaling, further revealing EBP50 regulated EGFR signaling via its interaction with EGFR. EGFR activation was associated with poor prognosis of CC patients. EBP50 could not predict the prognosis of all CC patients. However, after ruling out patients with egfr/ErbB mutation or copy number variation (CNV) and (chemo)radiation, which caused continuous EGFR activation and affected the prognosis of patients, respectively, EBP50 expression level exhibited the prognosis prediction ability, revealing EBP50 affected prognosis of CC patients via regulating EGFR signaling. In conclusion, EBP50 played an important role in CC cell proliferation and prognosis prediction of CC patients by interacting with EGFR and regulating EGFR signaling. EBP50 might be a potential precise therapeutic target or prognostic marker for CC patients.

Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular target for antineoplastic therapy.

Abstract 5486: Identification of novel candidate genes associated with sorafenib cytotoxicity

Abstract Background: Sorafenib is an oral multikinase inhibitor that decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. Novel pathways and targets of sorafenib activity remain to be identified, and no predictive biomarkers of sorafenib activity exist to help guide clinicians. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains were genomically characterized previously (PMID: 21623374). MEFs were treated with ten concentrations (0-300 μM) of sorafenib, incubated for 72 h, and then fixed and stained. Cytochrome C was immunolabeled in the fixed cells and imaged using an automated fluorescence microscope. An image analysis software assessed sorafenib-induced cytochrome C release from mitochondria. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the EMMA algorithm, was performed on cytochrome C EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values ≤ 1.0×10−7 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 72 h (17.2-44.5 μM) sorafenib incubations. We identified one peak (chromosome 9 from 51-52 Mb; p = 1.0×10−8), which reached genome-wide significance and significantly associated with cytochrome C release. From this peak, we have identified candidate genes that may underlie variability in sorafenib-induced cytochrome C release from mitochondria. A total of nine genes expressed in MEF cells at mRNA level are present in this QTL. Of particular interest, the identified locus contains Rdx, a gene that encodes radixin. Radixin is a component of the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) complex, and is conserved between humans and mice. In humans, radixin, as part of the EBP50 complex, has been shown to act as a tumor suppressor and to promote apoptosis in hepatocellular carcinoma by modulating β-catenin/E-cadherin (PMID: 23483729). It has also been shown to potentially enhance the metastatic potential of renal cell carcinoma (PMID: 20395446). Conclusions: Our high-throughput cellular genetics approach has detected robust interstrain cellular differences in sorafenib activity, and identified one region, which reached genome-wide significance, that potentially associates with sorafenib-induced cytochrome C release from mitochondria. Functional validation of Rdx and other promising candidates is currently ongoing. Citation Format: Daniel James Crona, Oscar Suzuki, O. Joseph Trask, Bethany Parks, Amber Frick, Timothy Wiltshire, Federico Innocenti. Identification of novel candidate genes associated with sorafenib cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2015-5486

Phosphorylation of Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) by Akt Promotes Stability and Mitogenic Function of S-phase Kinase-associated Protein-2 (Skp2)

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.

Abstract 338: Akt-Mediated Phosphorylation of EBP50 Regulates Skp2 Stability and Vascular Smooth Muscle Cell Proliferation

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) increases vascular smooth muscle cells (VSMC) proliferation and vascular remodeling following endoluminal vessel injury. Indeed, neointima formation is significantly reduced in EBP50 knockout mice. The mitogenic effect of EBP50 derives from the Akt-dependent stabilization of the S-phase kinase associating protein Skp2. However, the mechanisms by which Akt regulates EBP50-mediated cell proliferation are not known. We hypothesize that phosphorylation of EBP50 by Akt promotes its interaction with Skp2. This causes cytoplasmic re-localization and stabilization of Skp2 and VSMC proliferation. Using Akt inhibitors and siRNA inhibition of Skp2, we show that the mitogenic effect of EBP50 in VSMC is dependent on both Akt and Skp2. In vitro and in vivo phosphorylation assays show that Akt phosphorylates EBP50 at T156 in the second PDZ domain. Interestingly, NMR, fluorescence polarization and co-immunoprecipitation experiments show that Skp2 interacts with the first PDZ domain of EBP50, suggesting that T156 phosphorylation exerts allosteric effects on the interaction with Skp2. Expression of EBP50 in primary VSMC induces a re-localization of Skp2 from the nucleus to the cytoplasm, leading to its stabilization and VSMC proliferation. These effects were abrogated by mutations within the first PDZ domain or of T156 to Alanine. Notably, the interaction with EBP50 does not directly affect the function of Skp2 because mutation of the C-terminal Leu to Ala, which abrogates the interaction with EBP50, does not decrease the ability of Skp2 to degrade p21cip1 and p27kip1. Consistent with the observations in primary cells, the expression of Skp2 in injured femoral arteries was reduced in EBP50 knockout mice compared to WT mice. In conclusion, our studies identify a novel mechanism for the Akt-dependent regulation of cell proliferation by EBP50 during vascular remodeling. They indicate that inhibition of EBP50 may be useful in preventing neointima formation following angioplasty.

Clinical significance of EBP50 overexpression assessed by quantum dot analysis in gastric cancer

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a postsynaptic density-95/disc-large/zonula occludens-1 (PDZ) homologous domain-containing protein that is involved in cell signaling. EBP50 regulates cell apoptosis, proliferation and invasion. In the present study, the prognostic impact factor of EBP50 expression was evaluated using a quantum dot (QD)-based assay and immunohistochemistry (IHC). The EBP50 protein expression in gastric cancer (GC) tissues was evaluated using IHC and QD-IHC. The study included 101 patients with GC (29 females and 72 males, aged 24–81 years), diagnosed and treated at the General Surgery Department of Renmin Hospital of Wuhan University (Wuhan, China) between 2000 and 2005. The survival rate was calculated using the Kaplan-Meier method and log-rank tests. IHC and QD analyses of 101 GC tissue specimens revealed that EBP50-positive tumor cells were frequently present in GC. Increased EBP50 immunostaining was observed in 63 specimens (62.4%). The EBP50 expression levels were correlated with increased tumor size and the male gender. EBP50 was well distributed in the cytoplasm and nuclei of the GC cells. However, EBP50 protein expression exhibited no correlation with age, differentiation, stage or lymph node metastasis. There were no associations between the expression of EBP50 and the mean survival rates (IHC, 50.5 vs. 58.1 months, P>0.05; QD, 55.4 vs. 63.2 months, P>0.05). These findings suggest that EBP50 protein expression is not correlated with the prognosis of patients with GC. QD-IHC and IHC have similar advantages for the detection of EBP50 protein expression.

Tumor suppressor function of ezrin-radixin-moesin-binding phosphoprotein-50 through β-catenin/E-cadher in pathway in human hepatocellular cancer

To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) in hepatocellular carcinoma (HCC). Three human HCC cell lines, i.e., SM-MC7721, HepG2 and Hep3B, were used. We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50. Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells. In vitro cell proliferation was assessed with a Cell Counting Kit-8 (CCK-8) assay. Cell cycle distribution was assessed with flow cytometry. Invasion and migration ability of before and after the transfection were determined with a transwell assay. Cell apoptosis was demonstrated with Annexin V-FITC. The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice. The transfection efficiency was confirmed with Western blotting (1.36 ± 0.07 vs 0.81 ± 0.09, P < 0.01). The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells (P < 0.01). Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50 (61.3% ± 3.1% vs 54.0% ± 2.4%, P < 0.05). The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells (5.8 ± 0.8 vs 21.6 ± 1.3, P < 0.01). Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells (14.8% ± 2.7% vs 3.4% ± 1.3%, P < 0.05). The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells (0.28 ± 0.07 vs 0.56 ± 0.12, P < 0.05; 0.55 ± 0.08 vs 0.39 ± 0.07, P < 0.05). In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells (28.9 ± 7.2 vs 70.1 ± 7.2, P < 0.01). The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin, E-cadherin. EBP50 may serve as a potential therapeutic target in HCC.

Abstract 94: Scaffolding Protein EBP50 Regulates Vascular Smooth Muscle Nox1 Activity by Binding p47 phox

The NADPH oxidase (Nox) enzyme family is implicated in the pathogenesis of cardiovascular diseases (CVDs) including hypertension. In large vessel smooth muscle cells (SMCs), a unique hybrid Nox1 oxidase exists which propagates SMC dysfunction and hypertrophy. We recently demonstrated that these actions involve activation of Nox1 by hydrogen peroxide (H 2 O 2 ) leading to feed-forward production of reactive oxygen species and activation of apoptosis signal regulating kinase 1 (Ask1). Here we postulate that Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), a PDZ-domain scaffolding protein, is required for H 2 O 2 -mediated activation of SMC Nox1. In SMCs, knockdown of EBP50 by shRNA abolished superoxide (O 2 •- ) production in 50 μM H 2 O 2 -treated cells (37.7 ± 4.5, 79.9 ± 2.3 and 29.0 ± 3.5 x10 4 O 2 •- -mediated RLU for GFP plasmid vehicle-treated, GFP plasmid H 2 O 2 -treated and EBP50 shRNA plasmid H 2 O 2 -treated groups, respectively). Furthermore, H 2 O 2 -induced O 2 •- production was absent in SMCs isolated from EBP50 knockout (KO) mice (29.0 ± 2.7 vs 79.1 ± 2.4 x10 4 RLU for EBP50 KO vs wildtype after H 2 O 2 treatment). Intriguingly, p47 phox , an essential cytosolic subunit of the hybrid SMC Nox1 system, upon phosphorylation co-immunoprecipitated with EBP50 in COS-7 cells transfected with both proteins (Fig 1). Taken together, our data suggest that EBP50 promotes Nox activity by binding active p47 phox . These studies identify a novel partner in Nox1 activation and potentially provide new insights into the broader regulation of this enzyme family. Moreover, this added level of complexity could yield new therapeutic strategies to disrupt Nox in the treatment of hypertension and other CVDs.

The detection of EBP50 expression using quantum dot immunohistochemistry in pancreatic cancer tissue and down-regulated EBP50 effect on PC-2 cells

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a putative tumor suppressor that is correlated with many human cancers. However, the function of EBP50 in pancreatic cancer (PC) has not been described. In this paper, the EBP50 expression level in PC tissues was characterized. In vitro, the effects of EBP50 down-regulation by siRNA in PC-2 and MiaPaCa-2 cells were evaluated. In addition, possible mechanisms that mediate the influence of EBP50 were examined. Our results show that the EBP50 expression pattern changes during transformation as there is a loss of the normal apical membrane distribution and an ectopic cytoplasmic over-expression of EBP50; furthermore, the EBP50 expression level is subsequently decreased during malignant progression. Down-regulation of EBP50 promoted cancer cell proliferation, increased the colony-forming ability of cells and accelerated the G1-to-S progression. Additionally, the loss of EBP50 accentuated β-catenin activity, increased cyclin E and phosphorylated Rb expression, and attenuated p27 expression compared to control cells. Our results suggest that EBP50 may function as a potential tumor suppressor.

Abstract 362: Akt-Dependent Interaction with EBP50 Regulates Skp2 Localization and Stability

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout mice. We previously reported that the mitogenic effect of EBP50 is Akt-dependent and mediated by the S-phase kinase-associated protein 2 (Skp2), a component of the E3 ligase complex that degrades the cell cycle inhibitor p21cip1. The mechanisms by which Akt directly increases Skp2 stability are controversial. We hypothesize that EBP50 regulates Akt-dependent stabilization of Skp2. The C-terminal four amino acids of Skp2 (PSCL) are a canonical PDZ-binding sequence. Co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and Skp2. Mutation of the C-terminal Leu to Ala (L424A-Skp2) abrogated the interaction with EBP50. Akt potentiated the association between EBP50 and Skp2. Sequence analysis indicated the presence of an Akt phosphorylation motif (RPRLCT) in EBP50 between residues 151 and 156. Indeed, in vitro assays using purified EBP50 and constitutively active Akt showed that Akt phosphorylates EBP50. This effect was abrogated by a naturally occurring mutation R153Q in EBP50 that disrupts the Akt consensus sequence. EBP50 was expressed exclusively in the cytoplasm of VSMC, where it associated with the cytoskeleton. Cell fractionation and fluorescence microscopy studies showed that expression of EBP50, but not of R153Q, induced relocalization of Skp2 from the nucleus to the cytoplasm. EBP50 expression increased Skp2 stability and, consequently, VSMC proliferation. Collectively, these experiments show that Akt-mediated EBP50 phosphorylation increases the interaction with Skp2, regulates Skp2 cellular localization and increases Skp2 stability. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide the mechanistic basis for the effect of this scaffolding protein on VSMC proliferation and neointima formation.

Abstract 487: EBP50 Promotes Epidermal Growth Factor-Dependent Activation of Focal Adhesion Kinase and Vascular Smooth Muscle Cells Migration

The Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) is a scaffolding protein that regulates a variety of physiological functions. Previous studies showed that EBP50 promotes vascular smooth muscle cells (VSMC) proliferation and neointima formation following arterial injury. In this study the role of EBP50 on VSMC migration was characterized. VSMC migration was determined by wound-healing assays. The motility of primary VSMC isolated from EBP50 KO mice was significantly reduced compared to WT cells (25 um/h WT vs. 19 um/h KO, p&lt;0.05) resulting in a 50% reduction in wound coverage. Conversely, EBP50 expression increased VSMC migration. EBP50-null VSMC had fewer and larger focal adhesions than EBP50-expressing cells. Both assembly and disassembly of focal adhesion in response to epidermal growth factor (EGF) (determined by real time TIRF microscopy) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with both focal adhesion kinase (FAK) and EGF receptor (EGFR) and the formation of a complex containing both EGFR and FAK was increased by EBP50. Stimulation of VSMC with EGF induced FAK phosphorylation on Tyr925 in WT, but not in KO cell, whereas phosphorylation of Tyr397 was unaffected. Collectively, these observations indicate that EBP50 facilitates growth factors-dependent activation of FAK, and consequently, migration of VSMC. Therefore, in addition to increasing VSMC proliferation, EBP50 promotes neointima formation following arterial injury by increasing VSMC motility.

EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation

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.

EBP50 gene transfection promotes 5-fluorouracil-induced apoptosis in gastric cancer cells through Bax- and Bcl-2-triggered mitochondrial pathways

5-Fluorouracil (5-FU) plays an important role in the chemotherapy of advanced gastric cancer. However, genetic factors that affect therapeutic efficacy of 5-FU warrant further investigation. In the present study, using stable transfection of the ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) gene, we explored the genetic influences on 5-FU-induced apoptosis of human gastric cancer cells. Stable overexpression of the EBP50 gene was determined by reverse transcription polymerase chain reaction (RT-PCR) assay and western blot analysis. After treatment with 5-FU, cell growth activities in vitro were investigated by MTT assay. Cell apoptosis was evaluated by Hoechst 33258 staining and flow cytometry of Annexin V-FITC/PI staining. Compared with the BGC823 or BGC823/neo cells, EBP50 mRNA and protein levels in the BGC823/EBP50 cells (EBP50-transfected BGC823 cells) were markedly higher. Chemosensitivity and apoptosis rates of the BGC823/EBP50 cells were higher compared to the BGC823 and BGC823/neo cells following treatment with 5-FU. Stable overexpression of extrinsic EBP50 distinctly increases the 5-FU-induced apoptosis of gastric cancer cells, and is a novel strategy by which to improve the chemosensitivity of gastric cancer to 5-FU.

Abstract P110: EBP50 Regulates Vascular Smooth Muscle Cell Growth by Skp2-Mediated Degradation of p21/cip1

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-binding phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout (KO) mice. The molecular mechanisms underlying EBP50 actions in VSMC are unknown. Genetic ablation of EBP50 reduced VSMC proliferation and was associated with increased (5-fold) expression of the cell cycle inhibitor p21cip1 both in vessels and in primary cells. No differences in mRNA levels of p21cip1 were observed in WT and KO cells. However, the half-life of p21cip1 in KO VSMC was significantly longer than in WT VSMC (80 min vs. 45 min) and p21cip1 levels were similar in WT and KO VSMC treated with the proteasome inhibitor MG132. These observations suggest that EBP50 regulates post-translational degradation of p21cip1. The S-phase kinase-associated protein 2 (skp2) is a component of the E3 ligase complex that degrades p21cip1. The C-terminal four amino acids of skp2 (ProSerCysLeu) are a canonical PDZ-binding sequence. Indeed, co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and skp2. Mutation of the C-terminal Leu to Ala (L424A-skp2) abrogated the interaction with EBP50. Skp2 expression was significantly lower in KO than in WT cells and inhibition of EBP50 expression by an shRNA lentivirus decreased skp2 expression in WT cells. Moreover, expression of skp2, but not of the mutant L424A-skp2, in WT cells reduced p21cip1 levels. Therefore, EBP50 regulates both expression and activity of skp2 with attendant effects on p21cip1 and VSMC proliferation. Collectively, these experiments show that EBP50, by regulating skp2 and p21cip1 expression, controls VSMC proliferation and the progression of neointima formation. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide a mechanistic basis for the remarkable effect of this scaffolding protein on vascular remodeling.

Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells

Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis through EGFR activation.

Immunohistochemical analysis of ezrin-radixin-moesin-binding phosphoprotein 50 in prostatic adenocarcinoma

BackgroundEzrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is an adapter protein which has been shown to play an active role in a wide variety of cellular processes, including interactions with proteins related to both tumor suppression and oncogenesis. Here we use immunohistochemistry to evaluate EBP50's expression in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets).MethodsTissue microarrays were immunohistochemically stained for EBP50, with the staining intensities quantified using automated image analysis software. The data were statistically analyzed using one-way ANOVA with subsequent Tukey tests for multiple comparisons. Eleven cases of NDP, 37 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 103 cases of PCa, and 36 cases of Mets were analyzed in the microarrays.ResultsSpecimens of PCa and Mets had the lowest absolute staining for EBP50. Mets staining was significantly lower than NDP (p = 0.027), BPH (p = 0.012), NAC (p < 0.001), HGPIN (p < 0.001), and PCa (p = 0.006). Additionally, HGPIN staining was significantly higher than NAC (p < 0.009) and PCa (p < 0.001).ConclusionsTo our knowledge, this represents the first study comparing the immunohistochemical profiles of EBP50 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC and suggests that EBP50 expression is decreased in Mets. Given that PCa also had significantly higher expression than Mets, future studies are warranted to assess EBP50's potential as a prognostic biomarker for prostate cancer.

Abstract 5205: EBP50, a PDZ-containing protein, regulates EGFR-induced cell scattering and migration in human cancer biliary epithelial cells

Abstract Background: Cholangiocarcinoma is the second most frequent primary malignant tumor of the liver, accounting for 10-15% of all primary liver cancers. Cholangiocarcinoma grows at the expense of epithelial cells that line intra- and extra-hepatic bile ducts. The adaptor protein EBP50 (Ezrin-radixin-moesin Binding Phosphoprotein 50) binds and regulates transmembrane transporters and receptors including Epidermal Growth Factor Receptor (EGFR). EBP50 and EGFR are both expressed in the normal biliary epithelium. While EGFR participates in biliary carcinogenesis, implication of EBP50 in this cancer is unknown. The aim of the study was to determine the potential links between EBP50 and EGFR in human biliary carcinoma. Methods: In vitro studies were performed in the human biliary epithelial cell line, Mz-ChA-1, that retains epithelial phenotype and endogenous expression of EBP50 and EGFR. The role of EBP50 was examined using siRNA. Cell scattering and migration were evaluated using E-cadherin immunostaining and Boyden chamber, respectively. For in vivo study, 112 patients (mean age: 59.5±11 years, 55 females and 57 males) who underwent surgical resection for hilar (n=53) and peripheral (n=59) cholangiocarcinoma in one single institution were analyzed retrospectively. Immunohistochemistry on tissue microarrays was performed to assess EBP50 and EGFR expression. Results: EGF activates EGFR and the three main signaling pathways, ERK1/2, STAT3 and Akt, in Mz-ChA-1 biliary cancer cells. Inhibition of EBP50 in EGF-treated cells increases and maintains activation of EGFR, and of ERK1/2 and STAT3 but not Akt. Enhanced EGFR activation in response to EBP50 depletion confers sensitivity to EGFR tyrosine kinase inhibitor, gefitinib. Functionally, upon EGF, EGFR drives cell scattering by inducing disruption of adherens junctions. In addition, EGF activates formation of lamellipodia structures and therefore cell migration. Interestingly, inhibition of EBP50 in non EGF-stimulated cell has the same effects on cell scattering and migration as those observed with EGF alone. Cell dispersion due to EBP50 inhibition is abolished in the presence of gefitinib. Moreover, impact on cell scattering and migration is even greater when both EGF addition and EBP50 siRNA are combined. In vivo, delocalization of EBP50 normally expressed beneath the apical membrane is associated with EGFR expression in human biliary carcinoma tissues (p=0.002). Conclusion: These data provide evidence for an essential role of EBP50 in repressing EGFR-induced signaling pathway and migration. EBP50 at the membrane may be critical to confine cancer progression by preventing migration of biliary cancer cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5205.