Annexin A2 Protein Expression Research Articles

Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism

AbstractReduced plasma fibrinolysis has been identified as a potential risk factor for venous thromboembolism (VTE), but the role of cell surface fibrinolysis in VTE is unknown. The annexin A2/S100A10 complex serves as a coreceptor for plasminogen and tissue plasminogen activator (tPA), augmenting plasmin generation by 60-fold on the endothelial cell surface. Several studies in both mice and humans support the concept that A2 regulates fibrin homeostasis and intravascular thrombosis in vivo. Here, we examined A2 protein expression and function in 115 adult subjects with VTE and 87 healthy controls. Using peripheral blood mononuclear cells as a surrogate for endothelial cells, we found a 41% mean decrease in cell surface tPA-dependent fibrinolytic activity in subjects who had a positive personal and family history of VTE but tested negative for known inherited thrombophilias (ITs). A2 protein was reduced on average by 70% and messenger RNA levels by 30%, but neither decrease correlated with anticoagulant therapy. Neither cell A2 protein nor cell surface plasmin generation correlated with plasma-based clot lysis times, suggesting that the plasma and cell surface fibrinolytic systems operate independently of one another. These data suggest that reduced expression of annexin A2 protein is associated with cell surface hypofibrinolysis and may represent a novel risk factor for IT.

MicroRNA-206 regulates the epithelial-mesenchymal transition and inhibits the invasion and metastasis of prostate cancer cells by targeting Annexin A2.

The present study investigated the molecular mechanism by which microRNA-206 (miR-206) targets Annexin A2 (ANXA2) expression and inhibits the invasion and metastasis of prostatic cancer cells through regulation of the epithelial-mesenchymal transition (EMT). Using bioinformatics analysis, miR-206 was identified as the most promising candidate miRNA that targeted ANXA2. Prostate tissue specimens from 60 patients with prostate cancer, 30 patients with metastatic prostate cancer and 20 patients with benign prostatic hyperplasia (BPH) were examined for ANXA2 protein expression by immunohistochemistry and western blotting and for miR-206 expression by reverse transcription-quantitative polymerase chain reaction. Additionally, human prostate cancer PC-3 cells were transfected with miR-206 mimics, miR-206 inhibitors or a negative control sequence, and expression of ANXA2, E-cadherin and N-cadherin was detected by western blotting. Transwell assays were performed to determine the effect of altered miR-206 expression on the invasive behavior of PC-3 cells. Bioinformatics analysis predicted complementary binding between miR-206 and ANXA2 mRNA. ANXA2 protein expression was detected in a significantly higher proportion of BPH tissues (95%, 19/20) when compared with prostate cancer tissues (51.7%, 31/60; P<0.05). Similarly, ANXA2 was expressed in a significantly higher proportion of metastatic prostate cancer samples than that of prostate cancer samples (P<0.05). Expression of miR-206 was higher than that of ANXA2 in prostate cancer samples, but lower in BPH samples. Inhibition of miR-206 expression in PC-3 cells upregulated ANXA2 and E-cadherin protein expression levels, downregulated N-cadherin and vimentin, and promoted cell invasion in vitro. These data suggested that binding between miRNA-206 and ANXA2 mRNA may regulate EMT signaling, thereby suppressing the invasion and metastasis of prostatic cancer cells.

Biological Effect and Mechanism of the miR-23b-3p/ANXA2 Axis in Pancreatic Ductal Adenocarcinoma

Background/Aims: Accumulating evidence strongly suggests that microRNAs (miRNAs) modulate the expression of known tumor suppressor genes and oncogenes. In the present study, we found that the proliferation and invasion ability of pancreatic ductal adenocarcinoma (PDAC) cells were significantly suppressed by the overexpression of miR-23b-3p. In addition, there are miR-23b-3p binding sites in annexin A2 (ANXA2). Here, we investigated whether miR-23b-3p had an impact on the progression and metastasis of PDAC by targeting ANXA2. Methods: Cell proliferation, migration, and invasion, and cell cycle assays were performed to explore the effect of miR-23b-3p on various malignant phenotypes of pancreatic cancer cells. The size of tumors was observed following miR-23b-3p overexpression in an in vivo chick chorioallantoic membrane assay. Dual-luciferase reporter, quantitative real-time PCR, western blot, and immunohistochemical analyses were used to validate the relationship between miR-23b-3p and ANXA2 in vitro. Results: We observed that miR-23b-3p could bind specifically to the 3′ untranslated region of ANXA2 and inhibit its expression. MiR-23b-3p overexpression downregulated the expression of ANXA2 mRNA in PDAC cells and limited the size of tumors or even prevented tumor formation. In addition, there was a negative correlation between miR-23b-3p expression and ANXA2 protein expression in clinical specimens. Conclusion: MiR-23b-3p inhibits the development and progression of PDAC by regulating ANXA2 directly.

Relationship Between Circulating Tumor Cells and Annexin A2 in Early Breast Cancer Patients.

Annexin A2 (ANXA2) is a phospholipid-binding protein involved in fibrinolysis, cell proliferation, migration and metastatic dissemination. Circulating tumor cells (CTCs) are cells responsible for tumor dissemination and have a prognostic value in several types of cancers including breast cancer. Previously, we found correlation between CTCs and activation of coagulation. This study aimed to correlate CTCs with ANXA2 expression on CTCs, tumor cells and tumor associated stroma in primary breast cancer (PBC) patients. This prospective study included 101 PBC patients treated by primary surgery. CTCs were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay for the expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes [TWIST1, SNAI1, SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1)]. ANXA2 expression on CTCs was detected by qRT-PCR, while expression of ANXA2 in tumor specimen was evaluated by immunohistochemistry and expressed by a weighted histoscore, evaluating both the percentage of positive cells and the intensity of membrane and cytoplasmic staining. Results of hormone receptors, HER2 status, B-cell lymphoma 2 (bcl-2) protein expression and protein p53 were reported as either positive or negative on histopathology report without further quantification. CTCs were detected in 24.8% patients. Patients with epithelial CTCs had a significantly higher ANXA2 expression on CTCs than those of patients without CTCs (p=0.01). There was no association between CTCs and ANXA2 protein expression in tumor cells. However, patients, whom CTCs with EMT phenotype were detected in, had higher ANXA2 expression in tumor stroma when compared to those with absent EMT CTCs (p=0.04). Hormone-negative tumors had significantly higher ANXA2 expression in tumor cells compared to hormone-positive tumors (p=0.03). Similarly, tumors without bcl-2 protein expression had higher tumor levels of ANXA2 compared to tumor cells that were bcl-2 positive (p=0.05). ANXA2 stromal expression might play a key role in aggressive tumor phenotype associated with increased EMT CTCs release, however, other factors beyond ANXA2 are responsible for coagulation activation mediated by CTCs in breast cancer patients.

Curcumin-ER Prolonged Subcutaneous Delivery for the Treatment of Non-Small Cell Lung Cancer.

Non-small-cell lung cancer therapy is a challenge due to poor prognosis and low survival rate. There is an acute need for advanced therapies having higher drug efficacy, low immunogenicity and fewer side effects which will markedly improve patient compliance and quality of life of cancer patients. The purpose of this study was to develop a novel hybrid curcumin nanoformulation (Curcumin-ER) and evaluate the therapeutic efficacy of this formulation on a non-small cell lung cancer xenograft model. Use of curcumin, a natural anticancer agent, is majorly limited due to its poor aqueous solubility and hence it's low systemic bioavailability. In this paper, we carried out the nanoformulation of Curcumin-ER, optimized the formulation process and determined the anticancer effects of Curcumin-ER against human A549 non-small cell lung cancer using in vitro and in vivo studies. Xenograft tumors in nude mice were treated with 20 mg/kg subcutaneous injection of Curcumin-ER and liposomal curcumin (Lipocurc) twice a week for seven weeks. Results showed that tumor growth was suppressed by 52.1% by Curcumin-ER treatment and only 32.2% by Lipocurc compared to controls. Tumor sections were isolated from murine xenografts and histology and immunohistochemistry was performed. A decrease in expression of NFκB-p65 subunit and proliferation marker, Ki-67 was observed in treated tumors. In addition, a potent anti-angiogenic effect, characterized by reduced expression of annexin A2 protein, was observed in treated tumors. These results establish the effectiveness of Curcumin-ER in regressing human non-small cell lung cancer growth in the xenograft model using subcutaneous route of administration. The therapeutic efficacy of Curcumin-ER highlights the potential of this hybrid nanoformulation in treating patients with non-small cell lung cancer.

Expression and clinical significance of annexin A2 and human epididymis protein 4 in endometrial carcinoma.

BackgroundIt is well-known that the treatment and monitoring methods are limited for advanced stage of endometrial carcinoma. Biological molecules with expression changes during tumor progression become potential therapeutic targets for advanced stage endometrial carcinoma. Annexin A2 (ANXA2) has been reported to be overexpressed in recurrent endometrial carcinoma, and the expression of human epididymis protein 4 (HE4) is upregulated in endometrial carcinoma. What’s more, ANXA2 and HE4 interacted in ovarian cancer and promoted the malignant biological behavior. We speculated that their interaction may exist in endometrial carcinoma as well. We evaluated the expression and the correlation relationship of ANXA2 and HE4 in endometrial carcinoma.MethodsThe expression of ANXA2 and HE4 protein in 84 endometrial carcinoma, 30 endometrial atypical hyperplasia, and 18 normal endometrial tissue samples were then measured using an immunohistochemical assay in paraffin embedded endometrial tissues. The structural relationship between ANXA2 and HE4 was explored by immunoprecipitation and double immunofluorescent staining.ResultsANXA2 and HE4 co-localized in both endometrial tissues and endometrial carcinoma cells. ANXA2 and HE4 were expressed in 95.2 % and 85.7 % of the the endometrial carcinoma, respectively, which were significantly higher than normal endometrium (55.6 % and 16.7 %, both p < 0.05). The expression of ANXA2 and HE4 was significantly correlated with FIGO stage, degree of differentiation, myometrial invasion, and lymph node metastasis. ANXA2 was an independent risk factor for the prognosis of endometrial carcinoma (p < 0.05, hazard ratio [HR] = 8.004). The expression of ANXA2 and HE4 was positively correlated (Spearman correlation coefficient = 0.228, p < 0.05). HE4 was an independent factor for ANXA2 in multivariate linear regression model (p < 0.05).ConclusionWe revealed the co-localization of ANXA2 and HE4 in endometrial carcinoma. Expression levels of ANXA2 and HE4 were closely related to the malignant biological behavior of endometrial carcinoma, and ANXA2 was an independent risk factor for poor prognosis. The expression of ANXA2 and HE4 can affect each other.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0208-8) contains supplementary material, which is available to authorized users.

Abstract 5117: The expression of ERG oncoprotein and Annexin A2 show a strong inverse correlation in prostate cancers.

Abstract Introduction: Gene fusion of the TMPRSS2 promoter and ERG that gives rise to aberrant ERG expression is detected in over 50% of prostate cancers (CaP). ERG overexpression abrogates cell differentiation, promotes cell invasion and epithelial to mesenchymal transition, and additional Pten loss or AKT up-regulation enhances the transformation into well-differentiated prostate adenocarcinoma. Annexin A2 (ANXA2) is a calcium–dependent phospholipid–binding protein that regulates cell adhesion, cellular junction formation, actin remodeling and genesis of cell polarity. Evidence from a variety of malignancies suggests that ANXA2 is vital for promoting adhesion, migration and metastasis in cancer cells. In CaP, ANXA2 expression is lost or reduced, however, is increased in high grade tumors. Microarray gene expression studies have shown an inverse correlated ERG and ANXA2 expression. We examined the cancer associated functions of ERG and ANXA2 such as cell invasion, cell migration and cell polarity in CaP cell lines. Methods: ANXA2 and ERG cellular localization were monitored using fluorescence microscopy. The prostate specific transcriptional initiation site of ANXA2 was defined by 5’–RACE. Ch-IP Assay was used to show the recruitment of ERG to the ANXA2 promoter. Cases consisting of well differentiated and poorly differentiated CaP with and without TMPRSS2-ERG fusion were stained with ANXA2 and ERG antibodies to determine correlation in expression. Results: The inverse correlation between ERG and ANXA2 protein expression was confirmed by ERG knockdown experiments in VCaP cells. ERG overexpression in prostate adenocarcinoma is correlated with the loss ANXA2 expression in 90% of cases examined. A subset of ERG positive tumors exhibit small clusters of cells with clonal expression of ANXA2. We evaluated the patterns of ERG-ANXA2 expression against available clinical-pathological features. Conclusions: Our findings suggest that the expression of ANXA2 and its function in establishing cell polarity and cellular junctions may be disrupted by ERG overexpression through the transcriptional repression of ANXA2. We hypothesize that the reemergence of ANXA2 expression in advanced tumors reflects the accumulation of genetic hits that resulted in a more aggressive tumor with higher metastatic capacity. An IHC based assay with combined ERG and ANXA2 antibody cocktail could enhance the pathological evaluation of prostate tumors discern the re-expression of ANXA2 in ERG positive tumors and improve the prognosis and treatment of CaPs. This research was in part supported by the National Cancer Institute R01CA162383 grant to S. S. and USU–CPDR funds. Citation Format: Nick E. Griner, Shyh-Han Tan, Denise Young, Albert Dobi, Gyorgy Petrovics, Isabell Sesterhenn, Shiv Srivastava. The expression of ERG oncoprotein and Annexin A2 show a strong inverse correlation in prostate cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5117. doi:10.1158/1538-7445.AM2013-5117

Effect of shRNA Mediated Down-Regulation of Annexin A2 on Biological Behavior of Human Lung Adencarcinoma Cells A549

In the previous study, we found that Annexin A2 was significantly up-regulated in lung cancer and could induce related-antigen in lung cancer patients' serum. To further study the function of Annexin A2, the short hairpin RNA plasmid targeting Annexin A2 was constructed in vitro and transfected into human lung adencarcinoma A549 cells. Knocking down Annexin A2 expression by shRNA, the mRNA level of Annexin A2 was investigated by semi-quantitative RT-PCR. The expression of Annexin A2 protein was examined by Western Blotting and Immuocytochemistry. MTT assay and Transwell chamber model were used to evaluate proliferation and invasion of A549 cells in vitro. The concentration of matrix metalloproteinase-2 (MMP-2) and cathepsin B (CB) in the supernatant was evaluated by ELISA. At 48h after transfection, the expression of Annexin A2 mRNA and protein was down-regulated significantly, respectively (p < 0.05).The proliferation and invasion capability of A549 cells also decreased significantly (p < 0.05). The concentration of MMP-2 and CB was down-regulated obviously, respectively (p < 0.05). This study implies that Annexin A2 might play an important role in the progression and invasion of human lung cancer cells, and could promote progression of lung cancer by regulating the expression of MMP-2 and CB.

Annexin A2 expression in human esophageal squamous cell carcinoma and correlation with invasion and metastasis

Objective To explore Annexin A2 expression in human esophageal squamous cell carcinoma (ESCC) and investigate the correlation of Annexin A2 expression with invasion and metastasis of human ESCC. Methods From 2000 to 2008, specimens of Xinjiang medical University First Affiliated Hospital were collected. Pathologically confirmed ESCC surgical specimens were set as experimental group, and the corresponding tumor adjacent tissues located more than 5 cm far from ESCC center were set as control group. 22 fresh and 175 paraffin-embeded ESCC specimens with corresponding adjacent tissues were randomly collected as study samples. With qRT-PCR, Western-blot and immunohistochemistry, the expression of Annexin A2 were detected at the mRNA and protein level. The correlation between Annexin A2 expression and clinicopathological parameters was analyzed. Results In 22 pairs of fresh ESCC and corresponding tumor adjacent tissues, the expression of Annexin A2 at mRNA level was significantly higher in tumor adjacent tissues (0. 06 ± 0. 06) than that in ESCC (0. 02 ±0. 02) (P＜0.05 ). Annexin A2 expression at protein level was also significantly higher in tumor adjacent tissues (0.95±0. 42) than ESCC (0.81±0. 36) (P＜0.05). In 175 paraffin-embeded ESCC specimens and corresponding adjacent tissues, the positive rate of Annexin A2 protein expression was 82. 3% (144/175) of the ESCC samples, which was lower than corresponding tumor adjacent tissues 92. 0% (161/175)(P＜0. 05). In addition, Annexin A2 expression was correlated with lymphoid node metastasis (P＜0.05) and pathological differentiation in patients with ESCC (P＜0.05). However, there was no apparent correlation with gross type (P＞0. 05). Conclusion The low expression of Annexin A2 in ESCC maybe played a potential role in the carcinogenesis, invasion and metastasis. Key words: Esophageal neoplasms; Neoplasms, squamous cell; Annexin A2; Neoplasms invasion; Neoplasms metastasis