High Ezrin Expression Research Articles

A retrospective analysis of ezrin protein and mRNA expression in breast cancer: Ezrin expression is associated with patient survival and survival of patients with receptor-positive disease.

The cytoskeletal protein ezrin is upregulated in many cancer types and is strongly associated with poor patient outcome. While the clinical and prognostic value of ezrin has been previously evaluated in breast cancer, most studies to date have been conducted in smaller cohorts (less than 500 cases) or have focused on specific disease characteristics. The current study is the largest of its kind to evaluate ezrin both at the protein and mRNA levels in early-stage breast cancer patients using the Nottingham (n=1094) and METABRIC (n=1980) cohorts, respectively. High expression of ezrin was significantly associated with larger tumour size (p=0.027), higher tumour grade (p < 0.001), worse Nottingham Prognostic Index prognostic group (p=0.011) and HER2-positive status (p=0.001). High ezrin expression was significantly associated with adverse survival of breast cancer patients (p < 0.001) and remained associated with survival in multivariate Cox-regression analysis (p=0.018, hazard ratio (HR)=1.343, 95% confidence interval (CI)=1.051-1.716) when potentially confounding factors were included. High ezrin expression was significantly associated with adverse survival of patients whose tumours were categorised as receptor (oestrogen receptor (ER), progesterone receptor (PgR) or HER2) positive (p < 0.001) in comparison to those categorised as triple-negative breast cancer (p=0.889). High expression of ezrin mRNA (VIL2) in the METABRIC cohort was also significantly associated with adverse survival of breast cancer patients (p < 0.001). Retrospective analyses show that ezrin is an independent prognostic marker, with higher expression associated with shortened survival in receptor-positive (ER, PgR or HER2) patients. Ezrin expression is associated with more aggressive disease and may have clinical utility as a biomarker of patient prognosis in early-stage breast cancer.

Hypoxia and Ezrin Expression in Primary Melanoma Have High Prognostic Relevance.

Hypoxia affects tumor aggressiveness and activates pathways associated with epithelial mesenchymal transition (EMT) which are crucial for tumor progress. In this study, the correlation of hypoxia and EMT with sentinel lymph node status and tumor-specific survival was investigated in primary melanomas. CD34 for capillary count and Hypoxia inducible factor-1α (HIF-1α) as hypoxia indicators as well as Ezrin and L1-Cell Adhesion Molecule (L1CAM), both critical proteins contributing to EMT, were analyzed using immunohistochemistry in 49 melanoma patients with long follow-up (F/U, mean 110 months; range 12–263 months). We found a significant correlation between Breslow tumor thickness and Ezrin expression (p = 0.018). L1CAM expression in primary melanoma was significantly associated with HIF-1α expression (p < 0.0001) and sentinel lymph node metastasis (p = 0.011). Furthermore, low capillary count, reflecting hypoxic condition, was significantly associated with Ezrin expression (p = 0.047) and decreased tumor-specific survival (p = 0.035). In addition, patients with high Ezrin expression in their primary melanoma had a dramatic loss of life early in their F/U period (mean survival time 29 months; range 15–44 month). Our results highlight the relevance of Ezrin, L1CAM and HIF-1α as prognostic markers in melanoma patients. Additionally, we demonstrate that hypoxia in primary melanoma affects EMT and is at least partly responsible for early metastatic dissemination.

Prognostic significance of Ezrin expression in colorectal carcinoma

Objectives This study aimed to evaluate the immunohistochemical expression of Ezrin in various histopathological types of colorectal carcinoma, and to examine its association with various clinicopathological features and so evaluate its prognostic significance. Background Colorectal cancer is considered one of the most common cancers worldwide, it developed because of the cumulative effect of sequential genetic alterations. Thus, more extensive studies are needed to demonstrate the molecular mechanism of colorectal cancer initiation and progression. Ezrin gene has an important role in cell movement, migration, mitosis, and other physiological functions, and is involved in maintaining cell structure and cell motility. Its expression correlates with many human malignancies. Patients and methods Ezrin antibody immunostaining was performed using the avidin–biotin–peroxidase complex method in 204 cases of primary colorectal carcinomas, including 118 (57.8%) cases of adenocarcinoma, 56 (27.5%) cases of a mucoid carcinoma, and 30 (14.7%) cases of signet ring cell carcinoma. Results Cytoplasmic Ezrin expression was significantly higher in lymph-node-positive cases (P Conclusion High Ezrin expression is considered a poor prognostic factor for colorectal adenocarcinoma.

Comprehensive analysis of protein expression levels and phosphorylation levels in host skin in response to tick (Haemaphysalis longicornis) bite

Ticks are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens. The blood sucking of ticks causes varying degrees of damage to the skin of the host. Proteins related to immune regulation, vascular repair, and wound healing in mammalian skin respond to tick bites by regulating their expression and post-translational modifications to protect the skin from injury. Phosphorylation of proteins, as the most common post-translational modification of proteins, plays an important role in the rapid regulation of cell signal transduction, gene expression and cell cycle. To systematically explore the molecular regulatory mechanisms employed by mammalian skin to resist tick bites, larval, nymphal, and adult Haemaphysalis longicornis were used to bite the skin tissues of healthy rabbits in the present study. The quantitative proteomic technology data-independent acquisition was then carried out to investigate in depth the changes in protein expression and phosphorylation in rabbit skin after tick bite. The results showed that among the 4034 proteins and 1795 phosphorylated proteins identified, a total of 202 proteins and 435 phosphorylation sites were changed after H. longicornis bite. In order to provide convenience for sucking blood, active substances in the saliva of H. longicornis injected into the rabbit's skin can cause the expression level of trichohyalin and peptidyl arginine deiminase 3 in the skin of the host downregulate, which can make the host hair loss and regeneration disorders. At the same time, the active substances in saliva of the H. longicornis led to the phosphorylation of microtubule actin cross-linking factor 1 in the host's skin and further inactivation, so as to delay the healing of the host wound. In response to tick bites, the host skin promotes coagulation through high expression of fibrinogen and fibronectin, and vascular repair through high expression of integrin linked kinase and tenascin C, as well as accelerated phosphorylation of the phosphorylated protein Nck adaptor protein 1, and wound healing through high expression of ezrin and integrin. The upregulation of proteins such as coronin, NADPH oxidase, calnexin, and calreticulin and phosphorylation level of IL-4R in the host skin after the H. longicornis bite indicated that the immune response was playing an important defensive role in response to tick bites. Meanwhile, we found that the upregulated two lectins, mannose receptor C-type 1 and DC-SIGN, may serve as molecular makers to identify and monitor whether the skin is bitten by ticks. SignificanceHaemaphysalis longicornis are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens and are harmful to humans and livestock.The present study is the first quantitative proteomic study on protein expression levels in the rabbit skin after infection by H. longicornis. It is also the first quantitative phosphoproteomic study in the host skin infected by ticks. In this study, we found that tick bites cause the host hair loss and regeneration disorders. For resisting tick bite, the host activates the immune response and initiates vascular repair and wound-healing systems. In addition, some phosphorylated proteins promote host immunity and vascular repair. These results can help us further understand the defence mechanism of the host against tick bites, provide a basis for the development of an anti-tick vaccine, the development of anti-tick drugs, and the diagnosis of tick-borne diseases.

Ezrin and E-cadherin expression profile in cervical cytology: a prognostic marker for tumor progression in cervical cancer

BackgroundCervical cancer (CC) is the fourth cause of mortality by neoplasia in women worldwide. The use of immunomarkers is an alternative tool to complement currently used algorithms for detection of cancer, and to improve selection of therapeutic schemes. Aberrant expression of Ezrin and E-cadherin play an important role in tumor invasion. In this study we analyzed Ezrin and E-cadherin expression in liquid-based cervical cytology samples, and evaluated their potential use as prognostic immunomarkers.MethodsImmunocytochemical staining of Ezrin and E-cadherin was performed in cervical samples of 125 patients. The cytological or histological diagnostic was performed by Papanicolaou staining or H&E staining, respectively. HPV genotyping was determined using INNO-LIPA Genotyping Extra kit and the HPV physical status by in situ hybridization. Ezrin expression in HaCaT, HeLa and SiHa cell lines was determined by immunocytochemistry, immunofluorescence and Western blot.ResultsHigh Ezrin expression was observed in cervical cancer samples (70%), samples with multiple infection by HR-HPV (43%), and samples with integrated viral genome (47%). High Ezrin expression was associated with degree of SIL, viral genotype and physical status. In contrast, low E-cadherin expression was found in cervical cancer samples (95%), samples with multiple infection by HR-HPV/LR-HPV (87%) and integrated viral genome (72%). Low E-cadherin expression was associated with degree of SIL and viral genotype. Interestingly, Ezrin nuclear staining was associated with degree of SIL and viral genotype. High Ezrin expression, high percent of nuclear Ezrin and low E-cadherin expression behaved as risk factors for progression to HSIL and cervical cancer.ConclusionsEzrin and E-cadherin expression profile in cervical cytology samples could be a potential prognostic marker, useful for identifying cervical lesions with a high-risk of progression to cervical cancer.

Successful identification of a predictive biomarker for lymph node metastasis in colorectal cancer using a proteomic approach.

Colorectal cancer (CRC)-associated mortality is primarily caused by lymph node (LN) and distant metastasis, highlighting the need for biomarkers that predict LN metastasis and facilitate better therapeutic strategies. We used an Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based comparative proteomics approach to identify novel biomarkers for predicting LN metastasis in CRC patients. We analyzed five paired samples of CRC with or without LN metastasis, adjacent normal mucosa, and normal colon mucosa, and differentially expressed proteins were identified and subsequently validated at the protein and/or mRNA levels by immunohistochemistry and qRT-PCR, respectively. We identified 55 proteins specifically associated with LN metastasis, from which we selected ezrin for further analysis and functional assessment. Expression of ezrin at both the protein and mRNA levels was significantly higher in CRC tissues than in adjacent normal colonic mucosa. In univariate analysis, high ezrin expression was significantly associated with tumor progression and poor prognosis, which was consistent with our in vitro findings that ezrin promotes the metastatic capacity of CRC cells by enabling cell invasion and migration. In multivariate analysis, high levels of ezrin protein and mRNA in CRC samples were independent predictors of LN metastasis. Our data thus identify ezrin as a novel protein and mRNA biomarker for predicting LN metastasis in CRC patients.

Association of Ezrin expression with the progression and prognosis of gastrointestinal cancer: a meta-analysis.

BackgroundEzrin, a cytoskeletal protein, is involved in cell adhesion. Several studies have been performed to explore the association between Ezrin and gastrointestinal cancers, but the results are inconclusive. This meta-analysis aims to assess the prognostic value of Ezrin.Materials and Methods/FindingsPubMed and EMBASE were searched. Pooled hazard ratio (HR), odds ratio (OR) and 95% confidence intervals (CI) were utilized to evaluate the association between Ezrin expression and various clinical parameters. 2701 patients from 19 studies were included in the meta-analysis. For gastric cancer, Ezrin expression was associated with tumor grade (OR 2.32, 95% CI 1.53–3.52), TNM stage (OR 4.69, 95% CI 1.38–15.89), lymph node involvement (OR 3.96, 95% CI 1.47–10.70) and overall survival (HR 1.88, 95% CI 1.33–2.66). In colorectal cancer, Ezrin expression was associated with tumor grade (OR 3.94, 95% CI 2.10–3.78), TNM stage (OR 5.66, 95% CI 1.41–22.67), lymph node metastasis (OR 9.52, 95% CI 3.93–23.02), distant metastasis (OR 3.06, 95% CI 1.77–5.31), disease free survival (HR 2.48, 95% CI 1.44–4.28). For esophageal cancer, Ezrin expression was associated with lymph node metastasis (OR 2.07, 95% CI 1.00–4.28) and overall survival (HR 1.49, 95% CI 1.17–1.89).ConclusionsEzrin expression is significantly associated with tumor grade, TNM stage, and lymph node metastasis in gastric and colorectal cancers. For gastric cancers, Ezrin is useful in predicting distant metastasis. Survival data showed that high Ezrin expression is associated with poor prognosis in gastric, colorectal and esophageal cancers. Our findings suggest that Ezrin might be a potential biomarker in several gastrointestinal cancers.

Ezrin expression combined with MSI status in prognostication of stage II colorectal cancer.

Currently used factors predicting disease recurrence in stage II colorectal cancer patients are not optimal for risk stratification. Thus, new biomarkers are needed. In this study the applicability of ezrin protein expression together with MSI status and BRAF mutation status were tested in predicting disease outcome in stage II colorectal cancer. The study population consisted of 173 stage II colorectal cancer patients. Paraffin-embedded cancer tissue material from surgical specimens was used to construct tissue microarrays (TMAs) with next-generation technique. The TMA-slides were subjected to following immunohistochemical stainings: MLH1, MSH2, MSH6, PMS2, ezrin and anti-BRAF V600E antibody. The staining results were correlated with clinicopathological variables and survival. In categorical analysis, high ezrin protein expression correlated with poor disease-specific survival (p = 0.038). In univariate analysis patients having microsatellite instabile / low ezrin expression tumors had a significantly longer disease-specific survival than patients having microsatellite stable / high ezrin expression tumors (p = 0.007). In multivariate survival analysis, the presence of BRAF mutation was associated to poor overall survival (p = 0.028, HR 3.29, 95% CI1.14–9.54). High ezrin protein expression in patients with microsatellite stable tumors was linked to poor disease-specific survival (p = 0.01, HR 5.68, 95% CI 1.53–21.12). Ezrin protein expression is a promising biomarker in estimating the outcome of stage II colorectal cancer patients. When combined with microsatellite status its ability in predicting disease outcome is further improved.

Ezrin as a possible diagnostic and/or prognostic biomarker in mice lymphatic metastatic hepatocellular carcinoma in vivo.

Hepatocellular carcinoma (HCC) ranks in the top of cancers leading to death. Early diagnosis is the big challenge in the case of HCC. Our in vitro study showed that Ezrin expression in lymphatic metastasis hepatocellular carcinoma (LNM-HCC) was associated with the metastatic rate. Here we aim to evaluate Ezrin expression as diagnostic and/or prognostic biomarker of LNM-HCC in mice. Chinese inbred 615 mice, Hca-F and Hca-P cell lines were used in the study. Histological changes were determined by Hematoxylin and Eosin, while Ezrin expression was assessed by qRT-PCR, western blot, immunohistochemistry, and enzyme-linked immunosorbent assay. Ezrin expression in this study gives credit to our in vitro study which Ezrin expression was positively correlated with LNM-HCC and negatively with Annexin7 (A7) expression. The highest histological changes were observed in high metastatic primary/secondary tumors combined with high Ezrin expression. Ezrin and A7 are higher in total primary tumors than in total secondary tumors (P = 0.0001, P = 0.021), respectively. Ezrin expression was enhanced in Hca-P A7 down-regulated primary/secondary tumors (P = 0.004), whereas, Ezrin expression was suppressed in Hca-F A7 upregulated primary/secondary tumors. Serum ELISA indicated differential expression of Ezrin among the study groups (P ≤ 0.0001). Ezrin expression was higher in NC-Hca-F than NC-Hca-P (P ≤ 0.0001), suppressed in Hca-F A7 upregulation (P ≤ 0.0001) and in enhanced in Hca-P A7 down-regulation (P = 0.0001). In conclusion, Ezrin level may serve as a differential diagnostic and/or prognostic biomarker for high and low LNM-HCC and may be beneficial in the diagnosis of HCC disease. © 2017 BioFactors, 43(5):662-672, 2017.

Concomitant expression of ezrin and HER2 predicts distant metastasis and poor prognosis of patients with salivary gland carcinomas

Salivary gland carcinomas (SGCs) exhibit heterogeneous biological behaviors, including the formation of distant metastases, which is a critical event associated with poor prognosis. Ezrin, which is a member of the ezrin-radixin-moesin family of plasma membrane-cytoskeleton linker proteins, may provide a marker for metastasis and poor survival of patients with cancer. The aim of the present study was to investigate the relationship between ezrin expression and the expression of HER2, p53, and Ki-67 as well as clinicopathological factors in SGCs. For this purpose, we used immunohistochemistry to analyze the expression of these proteins in tissue microarrays prepared from formalin-fixed, paraffin-embedded primary tumor tissues of 221 patients with SGCs. Using receiver operating characteristic curves, we determined cut-off values of 30% and 5.0% for high expression of ezrin and Ki-67, respectively. High ezrin expression detected in samples from 63 (28.5%) patients with SGCs significantly correlated with male sex, high-grade histopathology, high Ki-67 labeling index, HER2 overexpression, aberrant expression of p53, and distant metastasis. Multivariate analysis demonstrated that high ezrin expression was an independent prognostic factor for shorter overall survival (hazard ratio, 2.11 [1.09-4.05]; P=.027). Furthermore, concomitant high expression of ezrin and HER2 overexpression correlated significantly with shorter disease-free survival and overall survival as well as a high incidence of distant metastasis (P<.001). These findings indicate that ezrin and HER2 expression in patients with SGCs represents a high-grade histopathological subtype that requires adjuvant therapy, including molecularly targeted therapies, to decrease the risk of subsequent metastasis.

Crumbs 3b promotes tight junctions in an ezrin-dependent manner in mammalian cells.

Crumbs 3 (CRB3) is a component of epithelial junctions, which has been implicated in apical-basal polarity, apical identity, apical stability, cell adhesion, and cell growth. CRB3 undergoes alternative splicing to yield two variants: CRB3a and CRB3b. Here, we describe novel data demonstrating that, as with previous studies on CRB3a, CRB3b also promotes the formation of tight junctions (TJs). However, significantly we demonstrate that the 4.1-ezrin-radixin-moesin-binding motifof CRB3b is required for CRB3b functionality and that ezrin binds to the FBM of CRB3b. Furthermore, we show that ezrin contributes to CRB3b functionality and the correct distribution of TJ proteins. We demonstrate that both CRB3 isoforms are required for the production of functionally mature TJs and also the localization of ezrin to the plasma membrane. Finally, we demonstrate that reduced CRB3b expression in head and neck squamous cell carcinoma (HNSCC) correlates with cytoplasmic ezrin, a biomarker for aggressive disease, and shows evidence that while CRB3a expression has no effect, low CRB3b and high cytoplasmic ezrin expression combined may be prognostic for HNSCC.

Ezrin Inhibition Up-regulates Stress Response Gene Expression

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.

Abstract P5-01-01: Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer

Abstract Lymph node (LN) metastasis is a key driver of recurrence and survival in breast cancer (BC) patients. However, the mechanisms of metastatic dissemination of tumour cells from LNs to distant sites and their predictors of response to systemic therapy remain poorly understood, mainly due to a lack of non-invasive in vivo imaging models. We have recently described ezrin, a pro-metastatic crosslinker protein, as a regulator of tumour lymphangiogenesis and metastasis in BC (Breast Cancer Res. 2014; 16(5): 438). Furthermore, we demonstrated significant association of high ezrin expression with lymphovascular invasion in a cohort (n=63) of premenopausal patients with invasive BC (p =0.024). These findings prompted us to examine the role of ezrin in migration and invasion of metastatic tumour cells in LNs and their response to ezrin-targeted therapy. Using a locally accrued LN positive patient cohort (n=94), we demonstrated a significant association between high ezrin levels and reduced recurrence-free survival (univariate Log-rank test, p=0.033), suggesting that ezrin is a potential predictor of relapse in LN positive BC. To address the mechanistic role of ezrin in LN metastasis, we developed a novel intravital imaging model using a lymphatic reporter transgenic mouse (B6-prox1-mOrange2-pA-BAC) to examine the response of tumour-draining LN to anti-ezrin systemic therapy in real time. Next, we tested the effects of a small molecule ezrin inhibitor (NSC668394) in vitro and observed significant suppression of ezrin activation (p-T567) and cancer cell invasive phenotype. Intravital imaging of inguinal LN metastases, derived from subcutaneously implanted breast adenocarcinoma E0771-LMV (lung metastatic variant) cells, demonstrated significant reduction in mobility and invasiveness (Mann Whitney, p&amp;lt;0.0001) of metastatic cells following systemic treatment with NSC668394 (0.5 mg/kg at 24h and 8h prior to imaging). Interestingly, LN metastases engagement by host T cell (CD3+) was notably increased, whereas T cell mobility was not affected by ezrin inhibition. Our findings present a novel non-invasive imaging model to study the LN metastasis response to anti-cancer therapy in real time, and provide new insight into the role of ezrin as a potential anti-metastatic target in BC. (Supported by CRS, CIHR, CBCF, BCAK, Queen's SRC). Citation Format: Ghaffari A, Hoskin V, Mullins G, Greer P, Kiefer F, Madarnas Y, SenGupta S, Elliott B. Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-01-01.

The impact of immunohistochemical staining with ezrin-carbonic anhydrase IX and neuropilin-2 on prognosis in patients with metastatic renal cell cancer receiving tyrosine kinase inhibitors.

The identification of prognostic factors in patients with renal cell carcinoma (RCC) represents an area of increasing interest. In this retrospective study, we evaluated the prognostic role of carbonic anhydrase-IX, ezrin, and neuropilin in metastatic RCC patients. The expression of several biomarkers were measured by immunohistochemistry (IHC) in 45 patients with advanced stage RCC treated with second-line tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factor (VEGF) after failure of interferon-alpha between January 2007 and June 2012. Kaplan-Meier curves and log-rank tests were used for analysis of progression-free survival (PFS) and overall survival (OS), and a multivariate Cox proportional hazard model was employed to identify factors with an independent effect on the survival. Age, ezrin and neuropilin-2 overexpression were found to be statistically significant factors (P < 0.05) for PFS in the univariate analysis. Ezrin and neuropilin-2 overexpression, hemoglobin and albumin level were statistically significant factors (P < 0.05) for OS in the univariate analysis. Multivariate analysis revealed that low expression of ezrin and neuropilin-2 was an independent prognostic factor for PFS and OS. The median PFS was 4 months for patients overexpressing neuropilin-2 versus 11 months for those with lower expression of neuropilin-2 (p = 0.033). The median OS was longer in patients with low levels of neuropilin-2 expression (26 months) compared to patients overexpressing neuropilin-2 (13 months) (p = 0.023). Increased expression of ezrin was associated with poor prognosis in patients treated with TKIs targeting VEGF (PFS, 3 vs 7 months; p = 0.012). High ezrin expression was associated with shorter OS (p = 0.009). This is the first study in the literature showing that neuropilin-2 and ezrin are related with prognosis in patients with advanced RCC.

Combined expression of ezrin and E-cadherin is associated with lymph node metastasis and poor prognosis in breast cancer.

Ezrin and E-cadherin have been known to play a role in tumor metastasis. However, the association between the expression of ezrin and E-cadherin in breast cancer patients remains unclear. In the present study, we investigated the expression of ezrin and E-cadherin in 275 breast cancer and 80 control patients with benign hyperplasia, using tissue microarray-based immunohistochemistry (IHC). Ezrin expression was higher, while that of E-cadherin was lower in breast cancer than in control samples. Ezrin expression was negatively correlated with E-cadherin expression in a subpopulation of breast cancer patients with a high expression of ezrin [ezrin(high)] and a low expression of E-cadherin [E-cad(low)]. The cytoplasmic expression of E-cadherin [E-cad(c)] occurred significantly more frequently in ezrin(high) breast cancer than in ezrin(low) breast cancer. The expression level of ezrin was significantly higher in breast cancer with E-cad(c) than that with a membrane expression of E-cadherin [E-cad(m)]. Ezrin(high) or E-cad(low) expression was more associated with lymph node metastasis, and shorter overall survival (OS) and disease-free survival (DFS) in breast cancer patients. E-cad(c) was more associated with lymph node metastasis and shorter OS and DFS compared with E-cad(m). The combined expression of ezrin(high) and E-cad(low) or ezrin(high) and ezrin(c) was more associated with lymph node metastasis and poor prognosis. In addition, the multivariate Cox regression analysis revealed that lymph node metastasis and ezrin(high) expression were independent prognostic factors for shorter OS and DFS in breast cancer patients. The results of the present study suggested that ezrin promotes breast cancer metastasis via the regulation of E-cadherin expression.

Ezrin protein overexpression predicts the poor prognosis of pancreatic ductal adenocarcinomas

Ezrin, a member of the ezrin/radixin/moesin (ERM) protein family, plays an important role in tumor metastasis. Accumulating studies demonstrated that a high expression level of human ezrin has been correlated with numerous human malignancies. This study was aimed to explore the clinicopathological significance of ezrin protein expression in pancreatic ductal adenocarcinomas (PDAC), and to further identify its role as a potential biomarker and therapeutic target of PDAC. Immunohistochemical (IHC) staining of ezrin protein was performed on 106 PDAC tissue samples and 37 adjacent and 21 normal pancreatic tissue samples. Additionally, localization of ezrin protein in Panc-1 PDAC cell line was observed using immunofluorescence (IF) staining. The correlation between ezrin overexpression and the clinicopathological features of PDAC was evaluated using Chi-square test, and differences in survival curves were analyzed using log-rank tests. In results, ezrin protein is widely distributed in the cytoplasm and membrane of PDAC cells by IHC and IF staining, but some cases showed a cell membrane staining pattern. The positive rate of ezrin protein expression was 82.1% (87/106) in PDAC, which was significantly higher than it in either adjacent pancreatic tissues (37.8%, 14/37) or normal pancreatic tissues (19.0%, 4/21). Overexpression of ezrin was closely related with larger tumor size, positive lymph node metastasis and advanced clinical stage. However, it was not correlated with patient age, gender, differentiation, Ki-67 expression index, and pancreas calcification point. Survival analysis showed that patients with ezrin high expression level had significantly lower overall survival rate than that with ezrin low expression level. Importantly, further analysis using a Cox proportional hazard regression model revealed that high ezrin expression emerged as a significant independent hazard factor for overall survival rates of patients with PDAC along with lymph node metastasis and TNM stage. In conclusion, ezrin protein played an important role in the progression of PDAC, and the overexpression of ezrin protein might be a useful prognostic marker of PDAC.

A novel role for ezrin in breast cancer angio/lymphangiogenesis.

IntroductionRecent evidence suggests that tumour lymphangiogenesis promotes lymph node metastasis, a major prognostic factor for survival of breast cancer patients. However, signaling mechanisms involved in tumour-induced lymphangiogenesis remain poorly understood. The expression of ezrin, a membrane cytoskeletal crosslinker and Src substrate, correlates with poor outcome in a diversity of cancers including breast. Furthermore, ezrin is essential in experimental invasion and metastasis models of breast cancer. Ezrin acts cooperatively with Src in the regulation of the Src-induced malignant phenotype and metastasis. However, it remains unclear if ezrin plays a role in Src-induced tumour angio/lymphangiogenesis.MethodsThe effects of ezrin knockdown and mutation on angio/lymphangiogenic potential of human MDA-MB-231 and mouse AC2M2 mammary carcinoma cell lines were examined in the presence of constitutively active or wild-type (WT) Src. In vitro assays using primary human lymphatic endothelial cells (hLEC), an ex vivo aortic ring assay, and in vivo tumour engraftment were utilized to assess angio/lymphangiogenic activity of cancer cells.ResultsEzrin-deficient cells expressing activated Src displayed significant reduction in endothelial cell branching in the aortic ring assay in addition to reduced hLEC migration, tube formation, and permeability compared to the controls. Intravital imaging and microvessel density (MVD) analysis of tumour xenografts revealed significant reductions in tumour-induced angio/lymphangiogenesis in ezrin-deficient cells when compared to the WT or activated Src-expressing cells. Moreover, syngeneic tumours derived from ezrin-deficient or Y477F ezrin-expressing (non-phosphorylatable by Src) AC2M2 cells further confirmed the xenograft results. Immunoblotting analysis provided a link between ezrin expression and a key angio/lymphangiogenesis signaling pathway by revealing that ezrin regulates Stat3 activation, VEGF-A/-C and IL-6 expression in breast cancer cell lines. Furthermore, high expression of ezrin in human breast tumours significantly correlated with elevated Src expression and the presence of lymphovascular invasion.ConclusionsThe results describe a novel function for ezrin in the regulation of tumour-induced angio/lymphangiogenesis promoted by Src in breast cancer. The combination of Src/ezrin might prove to be a beneficial prognostic/predictive biomarker for early-stage metastatic breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0438-2) contains supplementary material, which is available to authorized users.

DRP-1, ezrin and E-cadherin expression and the association with esophageal squamous cell carcinoma.

It has been shown that death-associated protein kinase (DAPK) family and E-cadherin play significant roles in the promotion of apoptosis and the suppression of cell adhesion and migration, and are involved in tumor metastasis. Ezrin, a cytoplasmic peripheral membrane protein, has been shown to interact with E-cadherin to participate in the metastasis of tumor cells. The present study aimed to investigate the expression of DRP-1 (a member of the DAPK family), ezrin and E-cadherin in esophageal squamous cell carcinoma (ESCC), and to analyze their association with clinicopathological factors in order to explore their potential in ESCC diagnosis. The expression of these genes was studied in tissue microarrays using in situ hybridization and immunohistochemistry methods in 76 specimens of ESCC and their paracancerous normal squamous epithelium tissues. Expression was statistically analyzed with regard to clinicopathological factors using χ2 and non-parametric tests. The expression level of DRP-1 was significantly different between the ESCC and paracancerous tissues (P<0.05). The expression level was correlated with the depth of invasion and lymph node metastasis (P<0.05). Abnormal E-cadherin expression was found to be associated with a high degree of cancer differentiation and lymph node metastasis (P<0.05). A positive correlation was observed between the expression of DRP-1 and E-cadherin (P<0.05). The expression of ezrin was found to be correlated with the depth of ESCC invasion, the degree of differentiation and lymph node metastasis (P<0.05). The high expression of ezrin has been previously shown to be correlated with the low or absent expression of E-cadherin. In conclusion, in ESCC, the expression levels of DRP-1, ezrin and E-cadherin were all reduced, and this reduction or absence of expression may have been attributed to ESCC tumorigenesis and progression. Simultaneous analyses of DRP-1, ezrin and E-cadherin expression levels would be useful to determine the malignancy and metastatic potential of ESCC, and these genes are consequently of potential use as biomarkers for the diagnosis and prognosis assessment of early-stage ESCC.

High expression of ezrin predicts poor prognosis in uterine cervical cancer

BackgroundEzrin, a member of the ezrin/radixin/moesin (ERM) protein family, plays a pivotal role in tumor invasion and metastasis. This study is aimed to investigate the clinicopathological significance of upregulated ezrin protein expression in uterine cervical cancers.MethodsImmunohistochemical staining of ezrin protein was performed on uterine cervical cancer specimens from 235 patients. For comparison, 239 cases of cervical intraepithelial neoplasia (CIN), 17 cases of cervical glandular intraepithelial neoplasia (CGIN) and 52 normal cervix samples were also included. qRT-PCR was performed on fresh tissues to detect ezrin mRNA expression levels. HPV infection statuses were genotyped by oligonucleotide microarray, and 10-year survival rates were calculated using the Kaplan-Meier method for 109 cervical cancer patients.ResultsApical membranous distribution of ezrin protein was only observed in normal cervical glands, while perinuclear staining was only observed in cervical cancers. Strong cytoplasmic and diffuse localization of ezrin were frequently seen in the cervical cancers compared with the normal counterparts. Furthermore, this strongly positive ezrin expression was significantly higher in cervical cancers than in CIN, CGIN, and normal cervical epithelia. Ezrin overexpression was closely related with poor differentiation, late stage, and lymph node metastasis. Additionally, ezrin overexpression was associated with lower 10-year survival rate for patients with early stage cervical cancer, but not for patients with advanced stage.ConclusionsAberrant localization and overexpression of ezrin might be an independent effective biomarker for prognostic evaluation of cervical cancers.

Ezrin Expression Is an Independent Prognostic Factor in Gastro-intestinal Cancers

BackgroundEzrin, a member of the ezrin–radixin–moesin (ERM) family of plasma membrane–cytoskeleton linker proteins, has been associated with metastatic behavior. MethodologyMicroarrayed pathological tissues of surgically resected colorectal cancer liver metastasis (CRLM) and whole tissue sections of cancer of the ampulla of Vater (CAV) were analyzed to determine ezrin expression levels and correlation with survival. The requirement of ezrin in invasive capability was assessed using in vitro assays. ResultsSurgically resected CAV showing a low ezrin score have a better 5-year disease-specific survival than those showing a high ezrin score (P < 0.0001). Similarly, high ezrin expression at the invasive front of CRLM resulted in poor disease-free survival (P = 0.05). Multivariate analysis demonstrated high ezrin expression to be an independent adverse prognostic factor for CAV (hazard ratio (HR) 15.22 (95 % confidence interval (CI) 1.98–117.03), P < 0.01) and CRLM (HR 6.42 (95 % CI 1.01–52.43), P = 0.05), among other clinically relevant variables such as lymph node metastasis (for CAV) and the presence of extrahepatic disease, large hepatic metastases (>5 cm), and close surgical resection margins (<5 mm) (all for CRLM). In vitro experiments indicated that ezrin expression was vital for cellular processes such as adhesive and invasive activity. SignificanceHigh ezrin expression indicates an adverse prognosis in primary CAV and CRLM.