Function Of Annexin A2 Research Articles

On the contribution of S100A10 and annexin A2 to plasminogen activation and oncogenesis: an enduring ambiguity.

Plasminogen receptors are becoming increasingly relevant in regulating many diseases such as cancer, stroke and inflammation. However, controversy has emerged concerning the putative role of some receptors, in particular annexin A2, in binding plasminogen. Several reports failed to account for the effects of annexin A2 on the stability and conformation of its binding partner S100A10. This has created an enduring ambiguity as to the actual function of annexin A2 in plasmin regulation. Supported by a long line of evidence, we conclude that S100A10, and not annexin A2, is the primary plasminogen receptor within the annexin A2-S100A10 complex and contributes to the plasmin-mediated effects that were originally ascribed to annexin A2.

Annexin A2 complexes with S100 proteins: structure, function and pharmacological manipulation.

Annexin A2 (AnxA2) was originally identified as a substrate of the pp60v-src oncoprotein in transformed chicken embryonic fibroblasts. It is an abundant protein that associates with biological membranes as well as the actin cytoskeleton, and has been implicated in intracellular vesicle fusion, the organization of membrane domains, lipid rafts and membrane-cytoskeleton contacts. In addition to an intracellular role, AnxA2 has been reported to participate in processes localized to the cell surface including extracellular protease regulation and cell-cell interactions. There are many reports showing that AnxA2 is differentially expressed between normal and malignant tissue and potentially involved in tumour progression. An important aspect of AnxA2 function relates to its interaction with small Ca2+-dependent adaptor proteins called S100 proteins, which is the topic of this review. The interaction between AnxA2 and S100A10 has been very well characterized historically; more recently, other S100 proteins have been shown to interact with AnxA2 as well. The biochemical evidence for the occurrence of these protein interactions will be discussed, as well as their function. Recent studies aiming to generate inhibitors of S100 protein interactions will be described and the potential of these inhibitors to further our understanding of AnxA2 S100 protein interactions will be discussed.

Inhibition of triple-negative and Herceptin-resistant breast cancer cell proliferation and migration by Annexin A2 antibodies.

Background:Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is abundantly present at the surface of triple-negative and Herceptin-resistant breast cancer cells. Interactions between cell-surface AnxA2 and tyrosine kinase receptors have an important role in the tumour microenvironment and act together to enhance tumour growth. The mechanism supporting this role is still unknown.Methods:The membrane function of AnxA2 was blocked by incubating cells with anti-AnxA2 antibodies. Western blotting, immunoprecipitation, immunofluorescence, 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), flow cytometry, Clonogenic, and wound-healing assays were performed in this study.Results:We demonstrate that AnxA2 interacts with epidermal growth factor receptor (EGFR) at the cell surface and has an important role in cancer cell proliferation and migration by modulating EGFR functions. Blocking AnxA2 function at the cell surface by anti-AnxA2 antibody suppressed the EGF-induced EGFR tyrosine phosphorylation and internalisation by blocking its homodimerisation. Furthermore, addition of AnxA2 antibody significantly inhibited the EGFR-dependent PI3K-AKT and Raf-MEK-ERK downstream pathways under both EGF-induced and basal growth conditions, resulting in lower cell proliferation and migration.Conclusions:These findings suggest that cell-surface AnxA2 has an important regulatory role in EGFR-mediated oncogenic processes by keeping EGFR signalling events in an activated state. Therefore, AnxA2 could potentially be used as a therapeutic target in triple-negative and Herceptin-resistant breast cancers.

Annexin A2 in renal cell carcinoma: Expression, function, and prognostic significance

ObjectiveRenal cell carcinoma (RCC) is the most lethal genitourinary cancer and intrinsically resistant to chemotherapy, radiotherapy, and hormone therapy. Annexin A2 (Anxa2) is a calcium-dependent phospholipid-binding protein found on various cell types that plays multiple roles in regulating cellular functions. In RCC, Anxa2 expression was correlated with tumor differentiation, clinical outcomes, and the metastatic potential; however, the underlying mechanisms remain obscure. This study investigated the role of Anxa2 in regulating tumorigenesis of RCC. Materials and methodsCommercial RCC tissue microarray arrays and a kidney cancer quantitative polymerase chain reaction array were used to examine Anxa2 by immunohistochemistry and real-time polymerase chain reaction analysis. Short hairpin (sh)RNA–based lentiviral system technology was used to evaluate the effects of manipulating Anxa2 expression on multiple malignant features of 2 RCC cell lines, A498 and 786-O, and its mechanisms. Results(1) The Anxa2 expression level was generally elevated to varying degrees in RCC tissues. In adjacent noncancerous tissues, Anxa2 was mainly expressed in glomeruli and slightly expressed in the cytoplasm of proximal tubules. (2) An increased Anxa2 expression level was found in tissues of clear cell RCC, papillary RCC, and chromophobe RCC, and it was prominently expressed in cancer cell membranes. In addition, the Anxa2 expression level was correlated with poor prognosis. (3) Silencing Anxa2 expression suppressed the abilities of cell migration and invasion, but cell proliferation was less affected. (4) Diminished Anxa2 expression caused alterations in the cell polarity, disrupted the formation of actin filaments, and reduced CXCR4 expression. (5) Inhibition of the Rho/Rock axis restored silencing of Anxa2-mediated suppression of cell motility. ConclusionsOverall, our study points out the regulatory function of Anxa2 in RCC cell motility and provides a molecular-based mechanism of Anxa2 positivity in the progression of RCC.

ANNEXIN A2 REGULATES ANGIOGENESIS AND INVASION PHENOTYPES OF MALIGNANT GLIOMA

BACKGROUND: Despite advances in multimodal therapy, patients with malignant glioma have a dismal prognosis. Aberrant angiogenesis and diffuse glioma cell invasion are major obstacles for effective treatment. Therefore, investigation of angiogenesis and invasion is essential for the development of a curative therapy. We established animal models that histologically mimic two invasive and angiogenic phenotypes of glioma, ie, angiogenesis-dependent invasion (J3T-1) and angiogenesis-independent invasion (J3T-2). In our previous study, comparative proteomic analysis showed that annexin A2 was more highly expressed in J3T-1 than in J3T-2. In this study, the function of annexin A2 in malignant glioma in relation to angiogenesis and invasion was investigated using these animal models. METHODS: Annexin A2-downregulated J3T-1 cells (J3T-1shA) and annexin A2-upregulated J3T-2 cells (J3T-2A) were generated by gene transfection. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistochemical staining of annexin A2 and vascular endothelial growth factor (VEGF) were performed on cultured cells. J3T-1, J3T-1shA, J3T-2, and J3T-2A brain tumors were established in athymic rats. Brain sections were evaluated histopathologically. Human glioblastoma specimens were stained for annexin A2, VEGF, and platelet-derived growth factor (PDGF). RESULTS: QRT-PCR and immunohistochemical staining of cultured cells revealed that expression of VEGF was upregulated in accordance with the expression of annexin A2 in J3T-1 and J3T-2A cells. J3T-1 tumors showed remarkable angiogenic activity and invasion around neovasculature, whereas J3T-1shA tumors, including J3T-2 tumors, showed no angiogenic activity, but diffuse single-cell infiltration of tumor cells into normal parenchyma. J3T-2A tumors were highly angiogenic and showed angiogenesis-dependent invasion. Human glioblastoma specimens revealed that annexin A2 was positive in clusters of tumor cells around dilated vessels (25/30 cases). The intensity of VEGF and PDGF staining corresponded to the expression of annexin A2 (correlation coefficients: annexin A2/VEGF: R = 0.73, P < 0.05; annexin A2/PDGF: R = 0.37, P < 0.05). CONCLUSIONS: Our results revealed that the phenotype of glioma invasion is closely related to angiogenesis, and annexin A2 is one of the factors that regulates angiogenesis and invasion phenotype of malignant glioma. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

Effect of Serine Phosphorylation and Ser25 Phospho-Mimicking Mutations on Nuclear Localisation and Ligand Interactions of Annexin A2

Annexin A2 (AnxA2) interacts with numerous ligands, including calcium, lipids, mRNAs and intracellular and extracellular proteins. Different post-translational modifications participate in the discrimination of the functions of AnxA2 by modulating its ligand interactions. Here, phospho-mimicking mutants (AnxA2-S25E and AnxA2-S25D) were employed to investigate the effects of Ser25 phosphorylation on the structure and function of AnxA2 by using AnxA2-S25A as a control. The overall α-helical structure of AnxA2 is not affected by the mutations, since the thermal stabilities and aggregation tendencies of the mutants differ only slightly from the wild-type (wt) protein. Unlike wt AnxA2, all mutants bind the anxA2 3′ untranslated region and β-γ-G-actin with high affinity in a Ca2+-independent manner. AnxA2-S25E is not targeted to the nucleus in transfected PC12 cells. In vitro phosphorylation of AnxA2 by protein kinase C increases its affinity to mRNA and inhibits its nuclear localisation, in accordance with the data obtained with the phospho-mimicking mutants. Ca2+-dependent binding of wt AnxA2 to phosphatidylinositol, phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate, as well as weaker but still Ca2+-dependent binding to phosphatidylserine and phosphatidylinositol-3,5-bisphosphate, was demonstrated by a protein–lipid overlay assay, whereas binding of AnxA2 to these lipids, as well as its binding to liposomes, is inhibited by the Ser25 mutations. Thus, introduction of a modification (mutation or phosphorylation) at Ser25 appears to induce a conformational change leading to increased accessibility of the mRNA- and G-actin-binding sites in domain IV independent of Ca2+ levels, while the Ca2+-dependent binding of AnxA2 to phospholipids is attenuated.

Abstract A052: Inhibition of triple-negative and Her-2 resistant breast cancer proliferation and migration by Annexin A2 antibodies

Abstract Introduction: The triple negative breast cancer (TNBC) phenotype, which lacks the presence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (ErbB-2 or Her-2), is highly aggressive and resistant to chemotherapy. In fact, some of the patients under hormone deprivation and/or Herceptin therapy acquire resistance. Therefore, there is an urgent need to identify new diagnostic as well as therapeutic markers for treatment of such patients. Herceptin, an immunotherapy directed against the Her-2 receptor, inhibits cancer growth and progression in Her-2 positive breast cancer by blocking the downstream survival pathways. In these cells however, the expression of epidermal growth factor receptor (EGFR) is significantly low. Interestingly, EGFR expression is significantly increased in triple negative and Her-2 resistant breast cancer thereby contributing to cancer growth and progression. Present studies were designed to investigate whether or not Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, regulated EGFR downstream signaling pathway and if the functions of EGFR can be inhibited by the AnxA2 antibody in TNBC and Herceptin resistant cancer cell lines. Methods: AnxA2 function at cell surface of the triple negative breast cancer cell line MDA-MB-231 and Her-2 resistant breast cancer cell line JIMT-1 was blocked incubating with AnxA2 antibody (2μg/ml) after 12h of serum starvation. The cells were treated with/without EGF (50ng/ml) for 20 min after 2h of antibody treatment. The cell lysate was analyzed for pEGFR and EGFR mediated downstream signaling by Western blotting. Cell migration was analyzed by scratch wound healing assay. Results: The results of the present study indicate that AnxA2 interacts with EGFR at the cell surface and plays an important role in the regulation of EGFR mediated downstream signaling. Treatment of MDA-MB-231 and JIMT-1 cells with AnxA2 antibody causes significant decrease in EGF-induced phosphorylation of EGFR at Y845 and Y1068 sites. Our results also demonstrate that blocking cell surface AnxA2 functions causes the downregulation of proteins such as pAKT, and pERK1/2 which are regulated by EGFR resulting in lower cell survival, proliferation, and migration. Conclusions: Our data indicate that association of AnxA2 with EGFR in the membrane domain plays a positive regulatory role in keeping EGFR signaling events in an activated state in both triple negative and Her-2 resistant breast cancer cells, thus making AnxA2 an important therapeutic target. Citation Format: Pankaj Chaudhary, Jamboor K. Vishwanatha. Inhibition of triple-negative and Her-2 resistant breast cancer proliferation and migration by Annexin A2 antibodies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A052.

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.

Tyrosine 23 Phosphorylation-Dependent Cell-Surface Localization of Annexin A2 Is Required for Invasion and Metastases of Pancreatic Cancer

The aggressiveness of pancreatic ductal adenocarcinoma (PDA) is characterized by its high metastatic potential and lack of effective therapies, which is the result of a lack of understanding of the mechanisms involved in promoting PDA metastases. We identified Annexin A2 (ANXA2), a member of the Annexin family of calcium-dependent phospholipid binding proteins, as a new molecule that promotes PDA invasion and metastases. We found ANXA2 to be a PDA-associated antigen recognized by post-treatment sera of patients who demonstrated prolonged survival following treatment with a PDA-specific vaccine. Cell surface ANXA2 increases with PDA development and progression. Knockdown of ANXA2 expression by RNA interference or blocking with anti-ANXA2 antibodies inhibits in vitro invasion of PDA cells. In addition, post-vaccination patient sera inhibits in vitro invasion of PDA cells, suggesting that therapeutic anti-ANXA2 antibodies are induced by the vaccine. Furthermore, cell-surface localization of ANXA2 is tyrosine 23 phosphorylation-dependent; and tyrosine 23 phosphorylation is required for PDA invasion. We demonstrated that tyrosine 23 phosphorylation resulting in surface expression of ANXA2 is required for TGFβ-induced, Rho-mediated epithelial-mesenchymal transition (EMT), linking the cellular function of ANXA2 which was previously shown to be associated with small GTPase-regulated cytoskeletal rearrangements, to the EMT process in PDA. Finally, using mouse PDA models, we showed that shRNA knock-down of ANXA2, a mutation at tyrosine 23, or anti-ANXA2 antibodies, inhibit PDA metastases and prolong mouse survival. Thus, ANXA2 is part of a novel molecular pathway underlying PDA metastases and a new target for development of PDA therapeutics.

Signal Transducer and Activator of Transcription 6 (STAT6) Is a Novel Interactor of Annexin A2 in Prostate Cancer Cells

Annexin A2 (AnxA2) is a multifunctional Ca(2+)-dependent phospholipid-binding protein, and its overexpression is implicated in malignant transformation of several cancers. In prostate cancer, however, the expression of AnxA2 is lost in prostate intraepithelial neoplasia and reappears in the high-grade tumors, suggesting a complex regulation of AnxA2 in the prostate microenvironment. Since a majority of the biological functions of AnxA2 are mediated by its interaction with other proteins, we performed a yeast two-hybrid assay to search for novel interactors of AnxA2. Our studies revealed that signal transducer and activator of transcription 6 (STAT6), a member of the STAT family of transcription factors, is a binding partner of AnxA2. We confirmed AnxA2-STAT6 interaction by in vitro co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies and demonstrated that AnxA2 interacts with phosphorylated STAT6. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that AnxA2 is associated with the STAT6 DNA-binding complex, and luciferase reporter assays demonstrated that AnxA2 upregulates the activity of STAT6. Upon interleukin-4 treatment, AnxA2 stabilizes the cytosolic levels of phosphorylated STAT6 and promotes its nuclear entry. These findings suggest that AnxA2-STAT6 interactions could have potential implications in prostate cancer progression. This report is the first to demonstrate the interaction of AnxA2 with STAT6 and suggests a possible mechanism by which AnxA2 contributes to the metastatic processes of prostate cancer.

Annexin A2 Binding to Endosomes and Functions in Endosomal Transport Are Regulated by Tyrosine 23 Phosphorylation

The phospholipid-binding annexin A2 (AnxA2) is known to play a role in the regulation of membrane and actin dynamics, in particular in the endocytic pathway. The protein is present on early endosomes, where it regulates membrane traffic, including the biogenesis of multivesicular transport intermediates destined for late endosomes. AnxA2 membrane association depends on the protein N terminus and membrane cholesterol but does not involve the AnxA2 ligand p11/S100A10. However, the precise mechanisms that control AnxA2 membrane association and function are not clear. In the present study, we have investigated the role of AnxA2 N-terminal phosphorylation in controlling association to endosomal membranes and functions. We found that endosomal AnxA2 was partially tyrosine-phosphorylated and that mutation of Tyr-23 to Ala (AnxA2Y23A), but not of Ser-25 to Ala, impaired AnxA2 endosome association. We then found that the AnxA2Y23A mutant was unable to bind endosomes in vivo, whereas a phospho-mimicking AnxA2 mutant (Y23D) showed efficient endosome binding capacity. Similarly, we found that AnxA2Y23D interacted more efficiently with liposomes in vitro when compared with AnxA2Y23A. To investigate the role of Tyr-23 in vivo, AnxA2 was knocked down with small interfering RNAs, and then cells were recomplemented with RNA interference-resistant forms of the protein. Using this strategy, we could show that AnxA2Y23D, but not AnxA2Y23A, could restore early-to-late endosome transport after AnxA2 depletion. We conclude that phosphorylation of Tyr-23 is essential for proper endosomal association and function of AnxA2, perhaps because it stabilizes membrane-associated protein via a conformational change.

Annexin A2 positively contributes to the malignant phenotype and secretion of IL‐6 in DU145 prostate cancer cells

Several groups, including ours, have reported that annexin A2 (ANXA2) expression is reduced in most prostate cancer (CaP). More recently, however, we reported that ANXA2 is expressed in some high-grade tumors, but the biologic consequence of this is currently unknown. To elucidate the function of ANXA2 in CaP, we reduced its expression in DU145 cells using shRNA and tested the impact on characteristics of malignancy. Reduction of ANXA2 suppressed anchorage-dependent and -independent cell growth without affecting invasiveness. Interestingly, interleukin-6 (IL-6) secretion was reduced concomitantly with the reduction of ANXA2 but independently of S100A10. IL-6 expression was restored when wild type but not mutant ANXA2 was reexpressed in these cells. In a retrospective study of radical prostatectomy specimens from patients with nonmetastatic CaP, 100% of patients with ANXA2-positive tumors (n = 4) had a biochemical relapse while only 50% of patients with ANXA2 negative tumors (n = 20) relapsed, suggesting that ANXA2 expression in prostate tumors may be predictive of biochemical relapse. Significant cytoplasmic staining of ANXA2 was detected in 3 of 4 ANXA2-positive tumors, whereas ANXA2 is localized to the plasma membrane in benign prostatic glands. These finding, taken together, suggests a possible mechanism whereby ANXA2 expression positively contributes to an aggressive phenotype in a subset of CaP and suggest that ANXA2 has markedly different functions depending on its cellular context. Finally, this is the first description of a role for ANXA2 in IL-6 expression, and ANXA2 represents a new therapeutic target for reducing IL-6 in high-grade prostate cancer.

Annexin A2 is a novel RNA-binding protein.

Annexin A2 (ANXA2) is a Ca(2+)-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (K(d) = 60 nM) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca(2+) (A(50%) = 10 microM). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca(2+)-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc.