Browsing by Subject "Annexin A2"
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Item CELL SURFACE TRANSLOCATION OF ANNEXIN A2 FACILITATES GLUTAMATE-INDUCED EXTRACELLULAR PROTEOLYSIS(2014-03) Maji, Sayantan; Vishwanatha, Jamboor K.; Valapala, MallikaNeurodegenerative diseases like age related macular degeneration (AMD) and Retinitis Pigmentosa (RP) are major causes of blindness affecting millions of people around the world. One of the major reasons of cell death observed in these diseases is the increased accumulation of glutamate, an excitatory amino acid. Unfortunately, the mechanisms behind glutamate induced toxicity are not yet known. Here we are investigating a possible role of a protein Annexin A2 (AnxA2) in glutamate induced toxicity. We found that glutamate causes increased membrane translocation of AnxA2. Increased membrane localization of AnxA2 and thereby its function can lead to the death of the eye cells leading to degenerative diseases like AMD and RP. The present study shows one of the possible mechanisms that can lead to glutamate induced cell death of the eye. Thus, using AnxA2 targeted therapy as an adjunctive therapy can lead to better and more efficient outcomes. Purpose (a): Glutamate-induced intracellular increase in Ca2+ levels leads to the hyper-activation of several normal Ca2+-mediated physiological processes including the activation of intracellular kinases, phosphatases, phospholipases and proteases which contribute to the degeneration of the retinal neurons as seen in many diseases including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Despite intensive research, the mechanisms that contribute to glutamate-induced cellular loss are yet to be elucidated. AnxA2, a Ca2+-dependent phospholipid binding protein serves as an extracellular proteolytic center by recruiting tissue plasminogen activator and plasminogen, and mediating localized generation of plasmin. We investigated whether AnxA2 plays a major role in glutamate induced neuronal excitotoxicity in a cone-photoreceptor cell line, 661W. Understanding the molecular mechanisms of glutamate-induced retinal degeneration can lead to the development of better therapeutic approaches for neurodegenerative diseases including AMD and RP. Our study provides new insights into one of the mechanisms that might contribute to glutamate-induced loss of photoreceptors in the retina. Methods (b): Ratiometric Ca2+ imaging and time lapse confocal microscopy were used to study glutamate-induced Ca2+ influx. EDTA eluates of 661W cells were immunoblotted to study the membrane translocation of endogenous as well as AnxA2-GFP in the presence or absence of different treatments. To determine whether glutamate induced membrane translocation of AnxA2 is dependent on the phosphorylation of the 23rd tyrosine residue or not, phosphomimetic and non-phosphomimetic variants were studied. Results (c): Glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N-terminus and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface translocated AnxA2 forms an active plasmin-generating complex and this activity can be neutralized by a hexapeptide directed against the N-terminus. Conclusions (d): These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. Thereby, targeting AnxA2 can be used as an adjunctive therapy in neurodegenerative diseases like AMD and RP.Item Higher Expression of Annexin A2 in Metastatic Bladder Urothelial Carcinoma Promotes Migration and Invasion(MDPI, 2022-11-27) Guo, Christina; Trivedi, Rucha; Tripathi, Amit K.; Nandy, Rajesh; Wagner, Diana C.; Narra, Kalyani; Chaudhary, PankajIn this study, we aim to evaluate the significance of AnxA2 in BLCA and establish its metastatic role in bladder cancer cells. Analysis of TCGA data showed that AnxA2 mRNA expression was significantly higher in BLCA tumors than in normal bladder tissues. High mRNA expression of AnxA2 in BLCA was significantly associated with high pathological grades and stages, non-papillary tumor histology, and poor overall survival (OS), progression-free survival (PFS), and diseases specific survival (DSS). Similarly, we found that AnxA2 expression was higher in bladder cancer cells derived from high-grade metastatic carcinoma than in cells derived from low-grade urothelial carcinoma. AnxA2 expression significantly mobilized to the surface of highly metastatic bladder cancer cells compared to cells derived from low-grade tumors and associated with high plasmin generation and AnxA2 secretion. In addition, the downregulation of AnxA2 cells significantly inhibited the proliferation, migration, and invasion in bladder cancer along with the reduction in proangiogenic factors and cytokines such as PDGF-BB, ANGPT1, ANGPT2, Tie-2, bFGF, GRO, IL-6, IL-8, and MMP-9. These findings suggest that AnxA2 could be a promising biomarker and therapeutic target for high-grade BLCA.Item Impact of Annexin A2 on virus life cycles(Elsevier B.V., 2024-05-04) Park, In-Woo; Fiadjoe, Hope K.; Chaudhary, PankajDue to the limited size of viral genomes, hijacking host machinery by the viruses taking place throughout the virus life cycle is inevitable for the survival and proliferation of the virus in the infected hosts. Recent reports indicated that Annexin A2 (AnxA2), a calcium- and lipid-binding cellular protein, plays an important role as a critical regulator in various steps of the virus life cycle. The multifarious AnxA2 functions in cells, such as adhesion, adsorption, endocytosis, exocytosis, cell proliferation and division, inflammation, cancer metastasis, angiogenesis, etc., are intimately related to the various clinical courses of viral infection. Ubiquitous expression of AnxA2 across multiple cell types indicates the broad range of susceptibility of diverse species of the virus to induce disparate viral disease in various tissues, and intracellular expression of AnxA2 in the cytoplasmic membrane, cytosol, and nucleus suggests the involvement of AnxA2 in the regulation of the different stages of various virus life cycles within host cells. However, it is yet unclear as to the molecular processes on how AnxA2 and the infected virus interplay to regulate virus life cycles and thereby the virus-associated disease courses, and hence elucidation of the molecular mechanisms on AnxA2-mediated virus life cycle will provide essential clues to develop therapeutics deterring viral disease.Item INHIBITION OF TRIPLE NEGATIVE AND HER-2 RESISTANT BREAST CANCER PROGRESSION BY ANNEXIN A2 ANTIBODIES(2013-04-12) Chaudhary, PankajPurpose: 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 another major tyrosine kinase receptor EGFR is significantly low. Interestingly, EGFR expression is significantly increased in Her-2 negative 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: Triple negative breast cancer cell line MDA-MB-231 and Her-2 resistant breast cancer cell line JIMT-1 were grown in complete DMEM and DMEM/F12 medium respectively, in a humidified incubator at 37C with 5% CO2. The AnxA2 function at cell surface was blocked incubating with AnxA2 antibody (2µg/ml) after 12 h of serum starvation. The cells were treated with/without EGF (50ng/ml) for 20 min after 2 h of antibody treatment. The cell lysate was analyzed for pEGFR and EGFR mediated downstream signaling by Western blotting. 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-mediated phosphorylation of EGFR at Y845 and Y1068 sites. In addition, treatment of cells with AnxA2 antibody decreases the ligand induced EGFR dimerization and internalization. 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: These studies indicate that association of AnxA2 with EGFR in the membrane domain might play a positive regulatory role in keeping EGFR signaling events in an activated state in triple negative and Her-2 resistant breast cancer thus making AnxA2 an important therapeutic target.Item MICRO-RNA MEDIATED DIFFERENTIAL EXPRESSION OF ANNEXIN A2 IN PROSTATE CANCER(2013-04-12) Maji, SayantanPurpose: Prostate cancer (PCa)develops through many defined stages: prostatic intraepithelial neoplasia (PIN), prostate cancer in situ, hormone-dependent and independent metastatic cancer. Annexin A2 (AnxA2), has been implicated in many cancer associated functions like plasminogen activation, actin-cytoskeletal rearrangement, cellular migration, adhesion and proliferation. Interestingly, AnxA2 is lost during PIN and early prostate cancer and reappears in metastatic prostate cancer, leading to worse clinical outcome. However the molecular mechanisms behind this differential regulation of AnxA2 are not yet understood. Our objective for this study is to identify the mechanism behind this differential regulation of AnxA2 in prostate cancer. Methods: As a proof of concept, miRNA maturation enzymes Drosha and Dicer were down regulated in hormone dependent PCa cells (LNCaP) by using siRNA, and AnxA2 mRNA expression was analyzed by PCR. Identification of the specific miRNAs that regulates AnxA2 expression in PCa was carried out by comparing the miRNA array profiling of normal and prostate cancer cell lines as well as in silico analysis. Identification of specific miRNA's was further confirmed by western blot analyses of the LNCaP and PC3 (metastatic prostate cancer), transfected with specific inhibitor of the miRNA and its mimic respectively. Migration and invasion assays were performed to study the effect of the specific miRNA on the prostate cancer cells. Results: Although LNCaP cells are null for AnxA2 expression, massive induction of AnxA2 mRNA expression by Drosha and Dicer knockdown confirmed the miRNA mediated regulation of AnxA2. The microarray study revealed three specific miRNAs. Out of these three miRNAs, miR-XYZ was first analyzed because of the high complimentary of its seed sequence to the coding region of the AnxA2 mRNA. Overexpression of AnxA2 in LNCaP and downregulation of AnxA2 PC3 cell lines upon treatment with miR-XYZ inhibitor and its mimic respectively were confirmed the Western blot. Transfection of miR-XYZ in PC3 cells resulted in a marked decrease in the migration and invasion of these cells as compared to control. Conclusions: These studies revealed that differential expression of AnxA2 is regulated by specific miRNA(s). Our results demonstrate that invasion and migration of PCa cells is significantly altered by modulating the expression of miR-XYZ. Thus miR-XYZ can be used as a potential diagnostic marker and as an adjuvant therapy in metastatic prostate cancer in the future.Item MIEN1 Drives Breast Cancer Invasion by Regulating Cytoskeletal-Focal Adhesions Dynamics(2015-05-01) Kpetemey, Marilyne F.; Vishwanatha, Jamboor K.; Clark, Abbot F.; Basu, AlakanandaIn the recent years, Migration and Invasion Enhancer 1(MIEN1) has emerged as a potential biomarker and a plausible target in breast cancer. Located in the 17q12-21 region of the human chromosome, next to the Her-2/neu loci, MIEN1 presents a robust expression in breast carcinomas; however is completely absent or low in the normal tissues. MIEN1 is post-translationally modified by geranyl-geranyl transferase-I (GgtaseI), which adds isoprenyl group to the carboxyl-terminal of the protein. Prenylated MIEN1 then associates with the inner leaflet of the plasma membrane and acts as an adaptor protein triggering downstream signaling through the Akt/NF-kB axis to regulate the expression of key proteases and angiogenic factors like MMP-9, uPA and VEGF. In migrating cells, MIEN1 enhances filopodium formation at the leading edge. Aside from its prenylation and redox-active motifs, MIEN1 also contains a canonical ITAM, reported to be associated with epithelial-to-mesenchymal transition. Although the role MIEN1 in cell migration and invasion is well known, the underlying molecular mechanisms remain elusive. Here, we show that MIEN1 interacts with Annexin A2, a cytoskeletal protein and a regulator of the plasminogen/plasmin system in breast cancer cells to increase migration and invasion. We confirmed that MIEN1 regulates actin dynamics by associating with cytoskeletal effectors in the lamellum. We also show that MIEN1 expression redirects breast tumor cell migration toward a collective migration. Our studies validate MIEN1-ITAM and CAAX as key motifs to MIEN1-induced functions. In conclusion, our findings confirm the role of MIEN1 in the remodeling of the actin cytoskeleton during motility. Furthermore it attests to previous findings suggesting that motility patterns depend on various environmental factors along with regulatory genes involved. Our study demonstrates an interesting example from cell biology where adaptor proteins regulate various signaling pathways and control cellular processes through protein-protein interactions.Item Phosphorlyation of Annexin A2 is essential for its association with exosomes and for migration, invasion and proliferation in triple negative breast cancer(2018-12) Desai, Priyanka P.; Vishwanatha, Jamboor K.; Basha, Riyaz; Chaudhary, PankajExosomes are membrane enclosed small vesicles that range from 40-120 nm in size and participate in cell-cell communication by transferring proteins to other cells. Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is present on the surface of the exosomes. AnxA2 phosphorylation plays an essential role in many physiological conditions by forming a heterotetrameric complex with p11 or S100A10 on the cell surface. We demonstrate here that the phosphorylation at Tyrosine (Tyr)-23 in the N-terminal region of AnxA2 is consequential for its association with the cell surface. This association increases the migratory, invasive and proliferative capacity of MDA-MB-231 triple negative breast cancer (TNBC) cells. An increase in cell surface AnxA2 further leads to a stronger association of AnxA2 with the exosomal surface. We also demonstrate that AnxA2 enriched exosomes promote proliferative and invasive characteristics of a different recipient cell [CAL (Centre Antoine Lacassagne) - 148]. These results demonstrate that Tyr23 phosphorylation of AnxA2 is pivotal for its association with exosomes and for imparting more malignant characteristics to the other breast cancer cells. Thus, AnxA2 could be used as a targeting approach for developing a treatment of TNBC.Item Role of Exosomal Annexin A2 in Angiogenesis and Breast Cancer Metastasis(2015-05-01) Maji, Sayantan; Vishwanatha, Jamboor K.; Clark, Abbot F.; Gryczynski, IgnacyEarly detection of cancer using circulating biomarkers is a realistic possibility with the discovery of exosomes. Cells under both physiological and pathological conditions secrete a wide array of membranous vesicles containing specific protein and RNA signatures. Exosomes, which are 40-100 nm in size, comprise a major portion of these vesicles. Exosomes play important roles in promoting tumor progression and metastasis, but the mechanisms by which they act are not yet understood. Annexin A2 (AnxA2) is a 36 kDa calcium dependent phospholipid-binding protein up-regulated in many cancer types that promotes tumorigenesis and angiogenesis. Although AnxA2 is highly expressed in exosomes, its function has never been characterized. In this study first we characterized exosomal AnxA2 (exo-AnxA2) expression in a breast cancer progression model. We found that exo-AnxA2 expression is significantly higher in malignant cells than normal and premetastatic cells. Next we explored the correlation and functionality of exo-AnxA2 in angiogenesis and breast cancer metastasis. In vitro and in vivo angiogenesis studies showed that exo-AnxA2 is an important mediator of angiogenesis and targeting the N-terminus of AnxA2 with a competitive hexapeptide greatly reduce the angiogenic effects induced by exo-AnxA2. To study the role of exo-AnxA2 in breast cancer metastasis we used MDA-MB-231 breast cancer cell line and its organ specific metastatic variants MDA-MB-831 (brain metastatic) and MDA-MB-4175 (lung metastatic) breast cancer cells. By using exosome priming in a mouse model, we show that priming with exosomes from metastatic breast cancer cells creates a favorable microenvironment for metastasis, and priming with exo-AnxA2 depleted exosomes leads to reduction of brain as well as lung metastasis. Detailed analysis of the signaling pathways revealed that p38MAPK/NF-κB and STAT3 pathways were up-regulated in the cancer exosomes primed animals than AnxA2 depleted exosome primed animals. These data demonstrate an important role for exo-AnxA2 in breast cancer pathogenesis. Finally, to validate whether exo-AnxA2 can be developed as s potential biomarker, we screened 50 breast cancer serum samples and 50 age matched control samples. Clinical analysis of the serum samples revealed that exo-AnxA2 is over expressed in breast cancer serum samples compared to normal samples. Detailed analysis of the exo-AnxA2 levels among breast cancer subtypes showed that more aggressive breast cancer subtype TNBC has higher exo-AnxA2 levels than HER2+ samples. In summary, our data suggest that exo-AnxA2 plays a major role in promoting angiogenesis and breast cancer metastasis. Further, breast cancer serum samples have higher exo-AnxA2 expression than control samples. Thus, exo-AnxA2 can be a potential diagnostic and prognostic marker in breast cancer patients to detect and monitor metastasis.Item Serum exosomal-annexin A2 is associated with African-American triple-negative breast cancer and promotes angiogenesis(BioMed Central Ltd., 2020-01-28) Chaudhary, Pankaj; Gibbs, Lee D.; Maji, Sayantan; Lewis, Cheryl M.; Suzuki, Sumihiro; Vishwanatha, Jamboor K.BACKGROUND: Limited information is available on biomarker(s) for triple-negative breast cancer (TNBC) that can address the higher incidence and aggressiveness of TNBC in African-American (AA) women. Our previous studies have demonstrated annexin A2 (AnxA2) association with exosomes which promotes angiogenesis and metastasis. Therefore, our goal was to examine the expression and function of exosomal-annexin A2 (exo-AnxA2) derived from the serum samples of breast cancer patients. METHODS: The expression of serum exo-AnxA2 and its association with clinicopathological features of the breast cancer patients were determined. The role of serum exo-AnxA2 to promote angiogenesis was determined by an in vivo Matrigel plug assay. RESULTS: Our results show that the expression of serum exo-AnxA2 in breast cancer patients (n = 169; 83.33 +/- 2.040 ng/mL, P < 0.0001) is high compared to non-cancer females (n = 68; 34.21 +/- 2.238 ng/mL). High expression of exo-AnxA2 levels in breast cancer was significantly associated with tumor grade (P < 0.0001), poor overall survival (hazard ratio (HR) 2.802; 95% confidence intervals (CI) = 1.030-7.620; P = 0.0353), and poor disease-free survival (HR 7.934; 95% CI = 1.778-35.398; P = 0.0301). The expression of serum exo-AnxA2 levels was significantly elevated in TNBC (n = 68; 109.1 +/- 2.905 ng/mL; P < 0.0001) in comparison to ER(+) (n = 50; 57.35 +/- 1.545 ng/mL), HER2(+) (n = 59; 78.25 +/- 1.146 ng/mL), and non-cancer females (n = 68; 34.21 +/- 2.238 ng/mL). Exo-AnxA2 showed diagnostic values with a maximum AUC as 1.000 for TNBC, 0.8304 for ER(+), and 0.9958 for HER2(+) compared to non-cancer females. The expression of serum exo-AnxA2 was significantly elevated in AA women with TNBC (n = 29; 118.9 +/- 4.086 ng/mL, P < 0.0001) in comparison to Caucasian-American TNBC (n = 27; 97.60 +/- 3.298 ng/mL) patients. Our in vivo results suggest a role of serum exo-AnxA2 in angiogenesis and its association with aggressiveness of TNBC in AA women. CONCLUSIONS: Our results demonstrated that the expression of serum exo-AnxA2 is high in AA women with TNBC and promotes angiogenesis. These findings suggest that exo-AnxA2 holds promise as a potential prognosticator of TNBC and may lead to an effective therapeutic option.Item STAT6 and Its Relationship with PSA and Annexin A2 in Human Prostate Cancer(2008-05-01) Roth, Cherice P.; Singh, Meharvan; Jones, Harlan P.; Sharma, RajendraRoth, Cherice, STAT6 and its relationship with PSA and Annexin A2 in Human Prostate Cancer. Master of Science (Biochemistry and Molecular Biology), May 2008, 49 pages, 13 illustrations, reference list, 54 titles. The increase of signal transducer and activator of transcription (STAT6) has been correlated with increased prostate tumor size as well as Gleason score. This molecule’s exact role in prostate cancer is still unknown. This research focused on the relationships of STAT6 in prostate specific antigen (PSA) expression as well as its novel interaction with annexin A2. These data show that STAT6 is involved in an alternate PSA expression pathway. It is also concluded that the interaction of STAT6 and annexin A2 increased the activated STAT6 (p-STAT) but not total STAT6. Chromatin immunoprecipitation also confirmed the novel protein-protein interaction between STAT6 and annexin A2 is nuclear.Item TETRANDRINE: A NOVEL CHEMOPREVENTIVE AGENT(2013-04-12) Gibbs, LeePurpose: The development and study of chemopreventive agents may be promising in combating aggressive behavior of triple-negative breast cancer. Tetrandrine, a bis-benzylisoquinoline alkaloid isolated from the root of Stephania tetrandra, is a calcium channel blocker used in Chinese medicine for the treatment of silicosis and arthritis. Studies have shown that tetrandrine also has anti-tumor and anti-growth activities. Our objective is to study the effects of tetrandrine on the localization of Annexin A2 (36 kDa calcium-dependent phospholipid binding protein) and determine the implication of this in the overall cell proliferation and cancer metastasis processes. We hypothesize that inhibition of calcium trafficking by tetrandrine will inhibit the migration, invasion and proliferation via attenuation of AnxA2 localization to the plasma membrane Methods: We have used Her-2 positive, triple-negative, and non-cancerous breast cell lines (MDA-MB-231, HCC70, BT474, SKBR3, and MCF-10A) to study the effects of tetrandrine. MTT assays were carried out to determine the effect of tetrandrine on cell viability. Additionally, cells were subjected to the Versene wash to analyze the effect of calcium depletion on the AnnXA2 localization in cells. Effects of tetrandrine on the expression and localization of Annexin A2 were also analyzed by the immunofluorescence and the Western blot analyses of the sub cellular fractions of the control and treated cells. Results: The viability of the cells at various concentrations of tetrandrine after incubation of 48 h measured by MTT assay showed IC50 values of 20uM, 25uM, 30uM, 40uM, and 75uM for HCC70, MDA MB231, SKBR3, BT474, and MCF10A respectively. Annexin A2 translocation to the membrane was analyzed by using a) versene (calcium ion chelating agent) washed cells, b) Western blot analyses of membrane and cytosolic extracts and c) Immunofluorescence of control and tetrandrine treated cells. Results of these studies indicated that treatment of breast cancer cells with tetrandrine did not show any significant effect on the Annx.A2 accumulation in plasma membrane of these cells when compared with the control cells. Conclusions: Our results suggest that tetrandrine inhibits the proliferation of breast cancer cells through mechanisms independent of Annexin A2. Significantly high IC50 value for MCF10A compared to the breast cancer cells indicates that this phyto-chemical may be used as a cancer chemopreventive agent.