Annexin A1 Knockdown Research Articles

EphB2 activates CREB-dependent expression of Annexin A1 to regulate dendritic spine morphogenesis

Dendritic spines are the postsynaptic structure to mediate signal transduction in neural circuitry, whose function and plasticity are regulated by organization of their molecular architecture and by the expression of target genes and proteins. EphB2, a member of the Eph receptor tyrosine kinase family, potentiates dendritic spine maturation through cytoskeleton reorganization and protein trafficking. However, the transcriptional mechanisms underlying prolonged activation of EphB2 signaling during dendritic spine morphogenesis are unknown. Herein, we performed transcriptional profiling by stimulating EphB2 signaling and identified differentially expressed genes implicated in pivotal roles at synapses. Notably, we characterized an F-actin binding protein, Annexin A1, whose expression was induced by EphB2 signaling; the promotor activity of its coding gene Anxa1 is regulated by the activity of CREB (cAMP-response element-binding protein). Knockdown of Annexin A1 led to a significant reduction of mature dendritic spines without an obvious deficit in the complexity of dendrites. Altogether, our findings suggest that EphB2-induced, CREB-dependent Annexin A1 expression plays a key role in regulating dendritic spine morphology.

Annexin A1 Is Required for Efficient Tumor Initiation and Cancer Stem Cell Maintenance in a Model of Human Breast Cancer.

Simple SummaryTriple-negative breast cancer (TNBC) has a poor outcome compared to the other major breast cancer subtypes and new therapies are needed. We sought to clarify the functions of a ubiquitous protein, Annexin A1, in the development and progression of TNBC. We found that Annexin A1 expression correlated with poor patient prognosis in basal-like breast tumors and also in the basal like-2 subset of TNBCs. Stable knockdown of Annexin A1 attenuated the growth of SUM149 xenografts, which model basal-like 2 tumors. In a polyoma middle T antigen-driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation, induced epithelial to mesenchymal transition and upregulated basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1− population containing putative tumor-initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in certain TNBC tumors.Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin A1 is an abundant multi-functional Ca2+ binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1− population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.

Knockdown of Annexin-A1 Inhibits Growth, Migration and Invasion of Glioma Cells by Suppressing the PI3K/Akt Signaling Pathway.

ANXA1, which can bind phospholipid in a calcium dependent manner, is reported to play a pivotal role in tumor progression. However, the role and mechanism of ANXA1 involved in the occurrence and development of malignant glioma are still not well studied. Therefore, we explored the effects of ANXA1 on normal astrocytes and glioma cell proliferation, apoptosis, migration and invasion and the underlying mechanisms. We found that ANXA1 was markedly up-regulated in glioma cell lines and glioma tissues. Down-regulation of ANXA1 inhibited normal astrocytes and glioma cell proliferation and induced the cell apoptosis, which suggested that the consequences of loss of Annexin 1 are not specific to the tumor cells. Furthermore, the siRNA-ANXA1 treatment significantly reduced tumor growth rate and tumor weight. Moreover, decreasing ANXA1 expression caused G2/M phase arrest by repressing expression levels of cdc25C, cdc2 and cyclin B1. Interestingly, ANXA1 did not affect the expressions of β-catenin, GSK-3β and NF-κB, the key signaling molecules associated with cancer progression. However, siRNA-ANXA1 was found to negatively regulate phosphorylation of AKT and the expression and activity of MMP2/-9. Finally, the decrease of cell proliferation and invasiveness induced by ANXA1 down-regulation was partially reversed by combined treatment with AKT agonist insulin-like growth factor-1 (IGF-1). Meanwhile, the inhibition of glioma cell proliferation and invasiveness induced by ANXA1 down-regulation was further enhanced by combined treatment with AKT inhibitor LY294002. In summary, these findings demonstrate that ANXA1 regulates proliferation, migration and invasion of glioma cells via PI3K/AKT signaling pathway.

AnnexinA1 expression is correlated with malignant potential of renal cell carcinoma.

To evaluate the expression of annexinA1 protein in patients with renal cell carcinoma. AnnexinA1 expression was examined in renal cell carcinoma specimens from 27 patients, and their disease-free survival was analyzed using the log-rank test. AnnexinA1 knockdown in the human renal cell carcinoma cell line Caki-1 was carried out, and its proliferation, invasion, motility and adhesion were compared with those of control cells. In 13 out of 27 patients, annexin A1 was highly expressed in the membrane of renal cell carcinoma tumor cells, whereas in the rest of the patients, annexinA1 expression was weak or negligible in the membrane of those cells. Patients with high annexinA1 expression had significantly poorer disease-free survival than those with weak or negligible annexinA1 expression (P=0.031). In the renal cell carcinoma cell line, annexinA1 knockdown cells showed significantly decreased proliferation, invasion, motility and adhesion relative to control cells, and expressed lower relative levels of membrane-type1 matrix metalloproteinase and hypoxia-inducible factor1-alpha transcripts, showing a potential pathway regulated by annexinA1. Annexin A1 is associated with renal cell carcinoma malignant potential and could serve as a marker of poor prognosis.

Knockdown of Annexin A1 Enhances Radioresistance and Inhibits Apoptosis in Nasopharyngeal Carcinoma.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma while radioresistance can hinder efficient treatment. To explore the role of annexin A1 and its potential mechanisms in radioresistance of nasopharyngeal carcinoma, human nasopharyngeal carcinoma cell line CNE2-sh annexin A1 (knockdown of annexin A1) and the control cell line CNE2-pLKO.1 were constituted and CNE2-sh annexin A1 xenograft mouse model was generated. The effect of annexin A1 knockdown on the growth of xenograft tumor after irradiation and radiation-induced DNA damage and repair was analyzed. The results of immunohistochemistry assays and Western blotting showed that the level of annexin A1 was significantly downregulated in the radioresistant nasopharyngeal carcinoma tissues or cell line compared to the radiosensitive nasopharyngeal carcinoma tissues or cell line. Knockdown of annexin A1 significantly promoted CNE2-sh annexin A1 xenograft tumor growth compared to the control groups after irradiation. Moreover, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays revealed that knockdown of annexin A1 significantly inhibited apoptosis in vivo compared to the control groups. We assessed the intracellular reactive oxygen species levels and the extent of radiation-induced DNA damage and repair using reactive oxygen species assay, comet assays, and immunohistochemistry assay. The results showed that knockdown of annexin A1 remarkedly reduced the intracellular reactive oxygen species levels, level of DNA double-strand breaks, and the phosphorylation level of H2AX and increased the accumulation of DNA-dependent protein kinase in nasopharyngeal carcinoma cells after irradiation. The findings suggest that knockdown of annexin A1 inhibits DNA damage via decreasing the generation of intracellular reactive oxygen species and the formation of γ-H2AX and promotes DNA repair via increasing DNA-dependent protein kinase activity and therefore improves the radioresistance in nasopharyngeal carcinoma cells. Together, our findings suggest that knockdown of annexin A1 promotes radioresistance in nasopharyngeal carcinoma and provides insights into therapeutic targets for nasopharyngeal carcinoma radiotherapy.

A novel anti-inflammatory mechanism of high density lipoprotein through up-regulating annexin A1 in vascular endothelial cells

High density lipoprotein (HDL) as well as annexin A1 have been reported to be associated with cardiovascular protection. However, the correlation between HDL and annexin A1 was still unknown. In this study, HDL increased endothelial annexin A1 and prevented the decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2±13.7% decrease for annexin A1 RNA interference; 78.7±16.3% decrease for anti-Annexin A1 antibody blocking; 11.2±6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte–endothelial cell adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.

Abstract 3906: Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling

Abstract Introduction: Annexin A1 is an anti-inflammatory protein reported to play a role in cell proliferation, apoptosis and to be deregulated in breast cancer. The role and regulation of annexin A1 in the biology and prognosis of breast cancer remain unclear because most of the studies did not consider specific differences between the different subtypes of breast cancer. Thus, in this study, we hypothesize that annexin A1 plays subtype-specific role in basal like breast cancer progression and metastasis. Methods: By mining The Cancer Genome Atlas-Breast Cancer Dataset (TCGA-BRCA) and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. Results: Our in-silico analysis of TCGA-BRCA dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer tumors than luminal and Her2/neu-positive breast cancer tumors. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway. Specifically, annexin A1 knockdown inhibited phosphorylation of mTOR and S6 (18-73% inhibition in pmTOR and 50-60% inhibition in pS6). But annexin A1 knockdown had no impact on the MAPK, c-Met, and EGFR pathways. In cell migration assays, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells which we found to be through suppression of MMP9. Conclusions: Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be due in part to the role of annexin A1 in activating mTOR-pS6 pathway. Citation Format: Kazunoshin Tachibana, Anjana Bhardwaj, Nivetha Ganesan, Kimal Rajapakshe, Constance T. Albarracin, Preethi H. Gunaratn, Cristian Coarfa, Isabelle Bedrosian. Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3906. doi:10.1158/1538-7445.AM2015-3906

Brain microvascular endothelium induced-annexin A1 secretion contributes to small cell lung cancer brain metastasis

Small cell lung cancer is the most aggressive histologic subtype of lung cancer, with a strong predilection for metastasizing to brain early. However, the cellular and molecular basis is poorly known. Here, we provided evidence to reveal the role of annexin A1 in small cell lung cancer metastasis to brain. Firstly, the elevated annexin A1 serum levels in small cell lung cancer patients were associated with brain metastasis. The levels of annexin A1 were also upregulated in NCI-H446 cells, a small cell lung cancer cell line, upon migration into the mice brain. More interestingly, annexin A1 was secreted by NCI-H446 cells in a time-dependent manner when co-culturing with human brain microvascular endothelial cells, which was identified with the detections of annexin A1 in the co-cultured cellular supernatants by ELISA and western blot. Further results showed that blockage of annexin A1 in the co-cultured cellular supernatants using a neutralized antibody significantly inhibited NCI-H446 cells adhesion to brain endothelium and its transendothelial migration. Conversely, the addition of Ac2-26, an annexin A1 mimic peptide, enhanced these effects. Furthermore, knockdown of annexin A1 in NCI-H446 cells prevented its transendothelial migration in vitro and metastasis to mice brain in vivo. Our data showed that small cell lung cancer cell in brain microvasculature microenvironment could express much more annexin A1 and release it outside, which facilitated small cell lung cancer cell to gain malignant properties of entry into brain. These findings provided a potential target for the management of SCLC brain metastasis.

Efficient Reovirus- and Measles Virus-Mediated Pore Expansion during Syncytium Formation Is Dependent on Annexin A1 and Intracellular Calcium

Orthoreovirus fusion-associated small transmembrane (FAST) proteins are dedicated cell-cell fusogens responsible for multinucleated syncytium formation and are virulence determinants of the fusogenic reoviruses. While numerous studies on the FAST proteins and enveloped-virus fusogens have delineated steps involved in membrane fusion and pore formation, little is known about the mechanics of pore expansion needed for syncytiogenesis. We now report that RNA interference (RNAi) knockdown of annexin A1 (AX1) expression dramatically reduced both reptilian reovirus p14 and measles virus F and H protein-mediated pore expansion during syncytiogenesis but had no effect on pore formation. A similar effect was obtained by chelating intracellular calcium, which dramatically decreased syncytiogenesis in the absence of detectable effects on p14-induced pore formation. Coimmunoprecipitation revealed calcium-dependent interaction between AX1 and p14 or measles virus F and H proteins, and fluorescence resonance energy transfer (FRET) demonstrated calcium-dependent p14-AX1 interactions in cellulo. Furthermore, antibody inhibition of extracellular AX1 had no effect on p14-induced syncytium formation but did impair cell-cell fusion mediated by the endogenous muscle cell fusion machinery in C2C12 mouse myoblasts. AX1 can therefore exert diverse, fusogen-specific effects on cell-cell fusion, functioning as an extracellular mediator of differentiation-dependent membrane fusion or as an intracellular promoter of postfusion pore expansion and syncytium formation following virus-mediated cell-cell fusion. Numerous enveloped viruses and nonenveloped fusogenic orthoreoviruses encode membrane fusion proteins that induce syncytium formation, which has been linked to viral pathogenicity. Considerable insights into the mechanisms of membrane fusion have been obtained, but processes that drive postfusion expansion of fusion pores to generate syncytia are poorly understood. This study identifies intracellular calcium and annexin A1 (AX1) as key factors required for efficient pore expansion during syncytium formation mediated by the reptilian reovirus p14 and measles virus F and H fusion protein complexes. Involvement of intracellular AX1 in syncytiogenesis directly correlates with a requirement for intracellular calcium in p14-AX1 interactions and pore expansion but not membrane fusion and pore formation. This is the first demonstration that intracellular AX1 is involved in pore expansion, which suggests that the AX1 pathway may be a common host cell response needed to resolve virus-induced cell-cell fusion pores.

Down-Regulation of Annexin A1 in the Urothelium Decreases Cell Survival After Bacterial Toxin Exposure

We examined the role of annexins in bladder urothelium. We characterized expression and distribution in normal bladders, biopsies from patients with bladder pain syndrome, cultured human urothelium and urothelial TEU-2 cells. Annexin expression in bladder layers was analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. We assessed cell survival after exposure to the pore forming bacterial toxin streptolysin O by microscopy and alamarBlue® assay. Bladder dome biopsies were obtained from 8 asymptomatic controls and 28 patients with symptoms of bladder pain syndrome. Annexin A1, A2, A5 and A6 were differentially distributed in bladder layers. Annexin A6 was abundant in detrusor smooth muscle and low in urothelium, while annexin A1 was the highest in urothelium. Annexin A2 was localized to the lateral membrane of umbrella cells but excluded from tight junctions. TEU-2 cell differentiation caused up-regulation of annexin A1 and A2 and down-regulation of annexin A6 mRNA. Mature urothelium dedifferentiation during culture caused the opposite effect, decreasing annexin A1 and increasing annexin A6. Annexin A2 influenced TEU-2 cell epithelial permeability. siRNA mediated knockdown of annexin A1 in TEU-2 cells caused significantly decreased cell survival after streptolysin O exposure. Annexin A1 was significantly reduced in biopsies from patients with bladder pain syndrome. Several annexins are expressed in human bladder and TEU-2 cells, in which levels are regulated during urothelial differentiation. Annexin A1 down-regulation in patients with bladder pain syndrome might decrease cell survival and contribute to compromised urothelial function.

Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin

Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA2), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA2 activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA2.

Cisplatin treatment leads to changes in nuclear protein and microRNA expression

Using a proteomic approach, we have previously shown that exposure to different concentrations of cisplatin during a 12-h period can lead to changes in nuclear protein expression and alternative splicing in HeLa cells. To further shed light on the DNA damage response (DDR) induced by cisplatin, we examined the nuclear proteome profiles of HeLa cells treated with 5μM cisplatin for different times (2, 12, and 24h). Two-dimensional electrophoresis (2-DE) identified 98 differentially expressed proteins in cisplatin-treated cells as compared to control cells. Among them, 54 spots (55%) were down-regulated and 44 spots (45%) were up-regulated. 51 spots were subjected to Matrix-assisted-laser-desorption-ionization Time-of-flight/time-of-flight Mass spectrometry (MALDI-TOF/TOF MS) identification, and 40 spots were identified. Among these, 22 proteins were located in nucleus. These proteins were involved in stress response, cell cycle and division, apoptosis, mRNA processing, transport, splicing and microRNA (miRNA) maturation. The changed expression of Annexin A1 and Lamin B1 were confirmed by Western blot. The role of Annexin A1 in the response to cisplatin-induced DNA damage was further analyzed, and it was shown that after Annexin A1 knockdown, cisplatin-induced DNA damage was significantly increased. In addition, the changed expression of several miRNAs was also observed by quantitative real-time PCR (qRT-PCR). Taken together, these data indicate that cisplatin-induced DDR is a complex process, and that those proteins identified by proteomics can lead to new directions for a better understanding of this process.

W10-O-001 Endothelial lipase deficiency increases plasma cholesterol but reduces susceptibility to atherosclerosis in apolipoprotein E-deficient mice

High density lipoprotein (HDL) as well as annexin A1 have been reported to be associated with cardiovascular protection. However, the correlation between HDL and annexin A1 was still unknown. In this study, HDL increased endothelial annexin A1 and prevented the decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2 ± 13.7% decrease for annexin A1 RNA interference; 78.7 ± 16.3% decrease for anti-Annexin A1 antibody blocking; 11.2 ± 6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte–endothelial cell adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.