ANXA2 is correlated with the molecular features and clinical prognosis of glioma, and acts as a potential marker of immunosuppression.

Recent studies have shown that ANXA2 is important in the development of many cancers, while its role in glioma-related immune response remains unclear. We aimed to comprehensively investigate its biological characteristics and clinical value in glioma. We analyzed 699 glioma samples from The Cancer Genome Atlas as training cohort and 325 samples from the Chinese Glioma Genome Atlas as validation cohort. All the statistical analyses and figures were generated with R. ANXA2 was overexpressed significantly in high-grade glioma, isocitrate dehydrogenase wild-type and mesenchymal-subtype glioma. ANXA2 was a special indicator of mesenchymal subtype. The survival analysis showed that highly-expressed ANXA2 was related to worse survival status as an independent factor of poor prognosis. Further gene ontology analysis showed that ANXA2 was mainly involved in immune response and inflammatory activities of glioma. Subsequent correlation analysis showed that ANXA2 was positively correlated with HCK, LCK, MHC II, STAT1 and interferon but negatively with IgG. Meanwhile, ANXA2 was positively related to the infiltration of tumor-related macrophages, regulatory T cells and myeloid-derived suppressor cells. Our study revealed that ANXA2 is a biomarker closely related to the malignant phenotype and poor prognosis of glioma, and plays an important role in immune response, inflammatory activity and immunosuppression.

Human colorectal cancer-associated carbohydrate antigen on annexin A2 protein.

Cancer-associated antigens are not only a good marker for monitoring cancer progression but are also useful for molecular target therapy. In this study, we aimed to generate a monoclonal antibody that preferentially reacts with colorectal cancer cells relative to noncancerous gland cells. We prepared antigens composed of HT-29 colorectal cancer cell lysates that were adsorbed by antibodies to sodium butyrate-induced enterocytically differentiated HT-29 cells. Subsequently, we generated a monoclonal antibody, designated 12G5A, which reacted with HT-29 colon cancer cells, but not with sodium butyrate-induced differentiated HT-29 cells. Immunohistochemical staining revealed 12G5A immunoreactivity in all 73 colon cancer tissue specimens examined at various degrees, but little or no immunoreactivity in noncancerous gland cells. Notably, high 12G5A immunoreactivity, which was determined as more than 50% of colon cancer cells intensively stained with 12G5A antibody, exhibited significantly higher association with a poor overall survival rate of patients with colorectal cancer (P = 0.0196) and unfavorable progression-free survival rate of patients with colorectal cancer (P = 0.0418). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, si-RNA silencing analysis, enzymatic deglycosylation, and tunicamycin treatment revealed that 12G5A recognized the glycosylated epitope on annexin A2 protein. Our findings indicate that 12G5A identified a cancer-associated glycosylation epitope on annexin A2, whose expression was related to unfavorable colorectal cancer behavior. KEY MESSAGE: • 12G5A monoclonal antibody recognized a colorectal cancer-associated epitope. • 12G5A antibody recognized the N-linked glycosylation epitope on annexin A2. • 12G5A immunoreactivity was related to unfavorable colorectal cancer behavior.

Autoantibodies to Annexin A2 and cerebral thrombosis: Insights from a mouse model.

INTRODUCTION: Antiphospholipid syndrome (APS) is an autoimmune disorder manifested by thromboembolic events, recurrent spontaneous abortions and elevated titers of circulating antiphospholipid antibodies. In addition, the presence of antiphospholipid antibodies seems to confer a fivefold higher risk for stroke or transient ischemic attack. Although the major antigen of APS is ß2 glycoprotein I, it is now well established that antiphospholipid antibodies are heterogeneous and bind to various targets. Recently, antibodies to Annexin A2 (ANXA2) have been reported in APS. This is of special interest since data indicated ANXA2 as a key player in fibrinolysis. Therefore, in the present study we assessed whether anti-ANXA2 antibodies play a pathological role in thrombosis associated disease. MATERIALS AND METHODS: Mice were induced to produce anti-ANXA2 antibodies by immunization with ANXA2 (iANXA2) and control mice were immunized with adjuvant only. A middle cerebral artery occlusion stroke model was applied to the mice. The outcome of stroke severity was assessed and compared between the two groups. RESULTS: Our results indicate that antibodies to ANXA2 lead to a more severe stroke as demonstrated by a significant larger stroke infarct volume (iANXA2 133.9 ± 3.3 mm3 and control 113.7 ± 7.4 mm3; p = 0.017) and a more severe neurological outcome (iANXA2 2.2 ± 0.2, and control 1.5 ± 0.18; p = 0.03). CONCLUSIONS: This study supports the hypothesis that auto-antibodies to ANXA2 are an independent risk factor for cerebral thrombosis. Consequently, we propose screening for anti-ANXA2 antibodies should be more widely used and patients that exhibit the manifestations of APS should be closely monitored by physicians.

Identification and characterization of antigen-specific CD4+ T cells targeting renally expressed antigens in human lupus nephritis with two independent methods.

In the search for anti-renal autoreactivity in human lupus nephritis, we stimulated blood-derived CD4+ T cells from patients with systemic lupus erythematosus with various kidney lysates. Although only minor responses were detectable, these experiments led to the development of a search algorithm that combined autoantibody association with human lupus nephritis and target gene expression in inflamed kidneys. Applying this algorithm, five potential T cell antigens were identified. Blood-derived CD4+ T cells were then stimulated with these antigens. The cells were magnetically enriched prior to measurement with flow cytometry to facilitate the detection of very rare autoantigen-specific cells. The detected responses were dominated by IFN-γ-producing CD4+ T cells. Additionally, IL-10-producing CD4+ T cells were found. In a next step, T cell reactivity to each single antigen was independently evaluated with T cell libraries and [3H]-thymidine incorporation assays. Here, Vimentin and Annexin A2 were identified as the main T cell targets. Finally, Vimentin reactive T cells were also found in the urine of three patients with active disease. Overall, our experiments show that antigen-specific CD4+ T cells targeting renally expressed antigens arise in human lupus nephritis and correlate with disease activity and are mainly of the Th1 subset.

Angiostrongylus cantonensis Galectin-1 interacts with Annexin A2 to impair the viability of macrophages via activating JNK pathway.

BACKGROUND: Angiostrongylus cantonensis can cause severe symptoms of central nervous system infections. In the host, this parasite localizes in the blood and cerebrospinal fluid, and its secreted components can impact immune responses. Our previous study demonstrated that immune responses were inhibited in A. cantonensis-infected mice immunized with Ac-Galectin-1 (AcGal-1). However, the mechanisms by which AcGal-1 regulates the immune responses remain unclear. Macrophages are innate immune cells that rapidly respond to infection. The direct impact of AcGal-1 on macrophages may affect the immune responses. METHODS: AcGal-1 protein was purified by nickel ion affinity chromatography. The effect of AcGal-1 on the apoptosis of macrophages was detected using CCK-8 assay, flow cytometry and western blot. Macrophage membrane proteins bound to AcGal-1 were obtained using the His-tag-based pull-down assay and identified via mass spectrometry. Co-localization of AcGal-1 and the macrophage membrane protein Annexin A2 was observed by immunofluorescence microscopy, and their interaction was validated by co-immunoprecipitation experiments. SiRNA-mediated knockdown of Annexin A2 was used to determine if AcGal-1-induced macrophage apoptosis required interaction with Annexin A2. The phosphorylation level of apoptotic signal pathway protein was detected by phospho-antibody microarray and western blot. RESULTS: Our study showed that AcGal-1 caused apoptosis of the macrophages. AcGal-1 increased the expression of apoptosis proteins caspase-3, caspase-9, Bax, but reduced the expression of anti-apoptosis protein Bcl-2. AcGal-1 interacted with the membrane protein Annexin A2, and knockdown of Annexin A2 expression increased Bcl-2 but decreased Bax levels in AcGal-1-treated cells. Moreover, AcGal-1 increased JNK phosphorylation and the inhibition of JNK phosphorylation in AcGal-1-treated cells decreased the expression of caspase-3, -9, Bax and almost restored Bcl-2 to the level observed in control cells. CONCLUSIONS: AcGal-1 can induce the apoptosis of macrophages by binding to Annexin A2 and activating JNK downstream the apoptotic signaling pathway.

Preclinical Activity of Embryonic Annexin A2-Specific Chimeric Antigen Receptor T Cells Against Ovarian Cancer.

Chimeric antigen receptors (CARs) have found clinical success in B cell malignancies, but a dearth of potential targets limits their wider clinical application, especially in solid tumours. Here, we describe the development of an anti-annexin A2 CAR, CAR(2448), derived from an antibody found to have activity against epithelial ovarian cancer cell lines. The spacer length of CAR(2448) was optimised based on in vitro cytotoxic activity against ovarian cancer (OC) cell lines via a real-time cytotoxicity assay. The longer spacer CAR(2448)L T cells exhibit significant effector activity, inducing inflammatory cytokine release and cytotoxicity against OC cell lines. Furthermore, CAR(2448)L-BBz T cells induced enhanced survival in an in vivo OC xenograft model and reduced tumour volume by 76.6%. Our preclinical studies of CAR(2448) suggest its potential for the unmet need of novel strategies for the treatment of ovarian cancer.

Anti-pancreatic tumor efficacy of a Listeria-based, Annexin A2-targeting immunotherapy in combination with anti-PD-1 antibodies.

BACKGROUND: Immune checkpoint inhibitors are not effective for pancreatic ductal adenocarcinoma (PDAC) as single agents. Vaccine therapy may sensitize PDACs to checkpoint inhibitor treatments. Annexin A2 (ANXA2) is a pro-metastasis protein, previously identified as a relevant PDAC antigen that is expressed by a GM-CSF-secreting allogenic whole pancreatic tumor cell vaccine (GVAX) to induce an anti-ANXA2 antibody response in patients with PDAC. We hypothesized that an ANXA2-targeting vaccine approach not only provokes an immune response but also mounts anti-tumor effects. METHODS: We developed a Listeria-based, ANXA2-targeting cancer immunotherapy (Lm-ANXA2) and investigated its effectiveness within two murine models of PDAC. RESULTS: We show that Lm-ANXA2 prolonged the survival in a transplant model of mouse PDACs. More importantly, priming with the Lm-ANXA2 treatment prior to administration of anti-PD-1 antibodies increased cure rates in the implanted PDAC model and resulted in objective tumor responses and prolonged survival in the genetically engineered spontaneous PDAC model. In tumors treated with Lm-ANXA2 followed by anti-PD-1 antibody, the T cells specific to ANXA2 had significantly increased INFγ expression. CONCLUSIONS: For the first time, a listeria vaccine-based immunotherapy was shown to be able to induce a tumor antigen-specific T cell response within the tumor microenvironment of a "cold" tumor such as PDAC and sensitize the tumor to checkpoint inhibitor therapy. Moreover, this combination immunotherapy led to objective tumor responses and survival benefit in the mice with spontaneously developed PDAC tumors. Therefore, our study supports developing Lm-ANXA2 as a therapeutic agent in combination with anti-PD-1 antibody for PDAC treatment.

Prevalence of anti-S100A10 antibodies in antiphospholipid syndrome patients.

INTRODUCTION: Annexin A2 (ANXA2), an endothelial cell receptor for plasminogen and tissue plasminogen activator, has been identified as a new autoantigen in antiphospholipid syndrome (APS). ANXA2 can exist as a monomer or a heterotetrameric complex with S100A10 protein. This S100A10 subunit also plays a pivotal role in the regulation of fibrinolysis. The aim of this study was to evaluate the prevalence of autoantibodies directed against S100A10 protein in patients with APS. METHODS: Patients with primary antiphospholipid syndrome (PAPS), patients with systemic lupus erythematosus (SLE) and patients with unexplained thrombosis were retrospectively included in this study. Patients were followed in the department of Internal Medicine of Amiens University Hospital, Amiens, France. IgG and IgM anti-S100A10 antibodies were detected in the serum of patients by enzyme-linked immunosorbent assay. The cut-off value for positivity was defined as 3 standard deviations above the mean optical density (OD) obtained in the sera of 116 healthy blood donors. RESULTS: The study group consisted of 116 healthy individuals and 106 patients: 42 APS patients (26 patients with PAPS and 16 patients with secondary SLE-related APS), 43 SLE patients without APS and 21 patients with unexplained thrombosis. The median age of APS patients, SLE patients without APS, patients with unexplained thrombosis and healthy individuals was 47, 38, 53 and 42 years, respectively. Anti-S100A10 antibodies were detected in 11.9% of APS patients and this prevalence was statistically higher than that observed in healthy individuals (1.7%) (p = 0.0148). Highest levels of anti-S100A10 were observed in the serum of one PAPS patient with venous thrombosis and one SLE patient with APS with a history of stroke and recurrent miscarriage. CONCLUSION: S100A10 protein, the binding partner of ANXA2, was identified as a target of autoantibodies in sera from patients with APS. Further studies involving a large cohort of APS patients are required to determine whether these antibodies could play a role in thrombogenic mechanisms of APS and to determine their diagnostic value in discriminating clinical subgroups of patients with APS, particularly those with seronegative APS.

Characterization of Ostertagia ostertagi annexin-like proteins at different developmental stages.

Ostertagiosis remains an economically important parasitic disease in cattle in the temperate regions of the world. Repeated exposures to Ostertagia ostertagi in calves cause significant pathology in the abomasum but elicit little protective immunity. The larvae use the host's gastric glands as a niche for development, where the parasite completes its parasitic stages, while in the gastric glands, the larvae must down-regulate the host inflammatory immune responses. Annexin (ANX) A1, commonly found in most eukaryotes, is heavily involved in controlling anti-inflammatory responses by binding receptors on leukocytes. We hypothesized, therefore, that parasite proteins of the ANX family may be involved in host-parasite interactions during ostertagiosis. BLASTN search with the bovine ANXA1 identified two families of Oos-ANX like proteins (Oos-ANXL), each of which was highly conserved at the genetic level and identical at the amino acid sequence level. Oos-ANXL-1 is encoded by two transcripts and Oos-ANXL-2 by 20 transcripts. The present study characterized one Oos-ANXL, representing the most abundant Oos-ANXL, which was further defined as Oost-ANXL-2.1. Oos-ANXL-2.1 with a coding sequence of 519 bp was PCR-amplified, cloned, and expressed. Oos-ANXL-2.1 was immunolocalized to both L3 and adult, but not L4. The staining appeared to be associated with the gut and hypodermis in L3, but it was specifically localized to the hypodermis in adult worms. Western blots detected three protein bands in parasite lysates using anti-recombinant Oos-ANXL-2.1 antibody. Integrated optical density for each of the 3 Oos-ANXL-2s or the total Oos-ANXL-2s detected by Western blots (P < 0.05) was higher in adult worms than in L3 or L4. The results indicate that the production of Oos-ANXL-2s is developmentally regulated and most abundant in the adult worm. This rather large family of proteins could be a potential vaccine target against O. ostertagi infection and warrants further investigation.

Complex Roles of Annexin A2 in Host Blood-Brain Barrier Invasion by Cryptococcus neoformans.

INTRODUCTION: Fungal transversal across the brain microvascular endothelial cells (BMECs) is the essential step for the development of cryptococcal meningoencephalitis. Annexin A2 (AnxA2) is an important signaling protein involved in several intracellular processes such as membrane trafficking, endocytosis, and exocytosis. AIM: To investigate the roles and mechanism of AnxA2 during cryptococcal transversal of BMECs. RESULTS: Cryptococcus neoformans infection initiated upregulation of AnxA2 in mouse BMECs. Blockade with anti-AnxA2 antibody led to a reduction in fungal transcytosis activity but no change in its adhesion efficiency. Intriguingly, AnxA2 depletion caused a significant increase in fungal association activity but had no effect on their transcytosis. AnxA2 suppression resulted in marked reduction in its partner protein S100A10, and S100A10 suppression in BMECs significantly reduced the cryptococcal transcytosis efficiency. Furthermore, AnxA2 dephosphorylation at Tyr23 and dephosphorylation of downstream cofilin were required for cryptococcal transversal of BMECs, both of which might be primarily involved in the association of C. neoformans with host cells. CONCLUSIONS: Our work indicated that AnxA2 played complex roles in traversal of C. neoformans across host BMECs, which might be dependent on downstream cofilin to inhibit fungal adhesion but rely on its partner S100A10 to promote cryptococcal transcytosis.

Cross-reactivity between annexin A2 and Beta-2-glycoprotein I in animal models of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) affects coagulation and the brain by autoimmune mechanisms. The major antigen in APS is beta-2-glycoprotein I (ß2-GPI) is known to complex with annexin A2 (ANXA2), and antibodies to ANXA2 have been described in APS. We measured these antibodies in mice with experimental APS (eAPS) induced by immunization with ß2-GPI. Sera of these mice reacted significantly with recombinant ANXA2 by enzyme-linked immunosorbent assay (ELISA) and the eAPS mice had significantly high levels of immunoglobulin G (IgG) in the brain by immunoblot assays compared to adjuvant immunized controls. Immunoprecipitation performed by mixing eAPS brain tissue with protein-G beads resulted in identification of two autoantigens unique to the eAPS group, one of which was ANXA2. In order to study more directly and methodically the specific role of anti-ANXA2 antibodies in APS, we immunized mice with ß2-GPI which contained no ANXA2 or with ANXA2 and measured antibodies to these proteins. Levels of antibodies to ANXA2 measured by ELISA were 0.72 ± 0.007 arbitrary units (a.u), 0.24 ± 0.03 and 0.02 ± 0.01 a.u for sera from ANXA2, ß2-GPI and control mice, respectively (p < 0.0001 and p = 0.037 for the comparison of the ANXA2 and ß2-GPI groups to the controls). Purified IgG from ß2-GPI sera did not show cross-binding with ANXA2. Antibodies to ß2-GPI and phospholipids were found in the ß2-GPI immunized group only. The present study suggests an immune response to the ß2-GPI-ANXA2 complex in eAPS and provides a novel ANXA2 immunization model which will serve to study the role of ANXA2 antibodies in of APS.

Antibodies directed against annexin A2 and obstetric morbidity.

Acquired and inherited thrombophilia have both been reported to be associated with an increased risk of obstetric complications in early or later stages of pregnancy. Annexin A2 (ANXA2) is strongly expressed in vascular and placental tissues and plays a crucial role in fibrinolysis. The aim of the present study was to evaluate the prevalence of antibodies directed against ANXA2 in patients with recurrent miscarriage or obstetric complications. Anti-ANXA2 antibodies (aANXA2) were detected by ELISA in the sera from 46 women with obstetric morbidity, mainly recurrent miscarriage. The cut-off value for positivity was defined as 3 standard deviations above the mean optical density (OD) obtained in the sera from 42 female blood donors. The prevalence of aANXA2 in patients and healthy individuals was 15.2% and 2.3%, respectively. A statistically significant difference was observed between the 2 groups in terms of aANXA2 IgG titers (p=0.01). The highest aANXA2 levels were observed in sera from 2 patients with recurrent miscarriage and one patient with preeclampsia. aANXA2 could play a role in thrombotic mechanisms leading to recurrent pregnancy loss and placental vascular disease. Further studies are needed to determine whether ANXA2 is critical for maintenance of placental integrity.

Annexin A2 Modulates ROS and Impacts Inflammatory Response via IL-17 Signaling in Polymicrobial Sepsis Mice.

Sepsis is a progressive disease manifesting excessive inflammatory responses, severe tissue injury, organ dysfunction, and, ultimately, mortality. Since currently, there are limited therapeutic options for this disease, further understanding the molecular pathogenesis of sepsis may help develop effective treatments. Here we identify a novel role for Annexin A2 (AnxA2), a multi-compartmental protein, in inhibiting pro-inflammatory response by regulating reactive oxygen species (ROS) and IL-17 signaling during sepsis. In cecal ligation and puncture (CLP) sepsis models, anxa2-/- mice manifested increased pro-inflammatory cytokines and neutrophil infiltration, but decreased bacterial clearance and animal survival. In addition, AnxA2 deficiency led to intensified ROS and IL-17A. Using site directed mutagenesis, we uncovered that cysteine 9 of AnxA2 was the most important aa (site) for regulation of ROS levels. Furthermore, ROS appears to be responsible for elevated IL-17A levels and subsequently exaggerated inflammatory response. Depletion of IL-17 via CRISPR/Cas9 KO strategy down-regulated inflammation and conferred protection against sepsis in anxa2-/- mice. Our findings reveal a previously undemonstrated function for AnxA2 in inflammatory response in polymicrobial sepsis models via an AnxA2-ROS-IL-17 axis, providing insight into the regulation of pathophysiology of sepsis.

Annexin A2, autoimmunity, anxiety and depression.

OBJECTIVES: Antiphospholipid syndrome (APS) is associated with neurological manifestations and one of the novel autoantigens associated with this disease is Annexin A2 (ANXA2). In this work we have examined the effect of high levels of autoantibodies to ANXA2 on the brain in a mouse model. METHODS: Recombinant ANXA2 emulsified in adjuvant was used to immunize mice while mice immunized with adjuvant only served as controls. At peak antibody levels the animal underwent behavioral and cognitive tests and their brains were examined for ANXA2 immunoglobulin G (IgG) and expression of ANXA2 and the closely linked protein p11. RESULTS: Very high levels of anti-ANXA2 antibodies (Abs) were associated with reduced anxiety in the open field 13.14% ± 0.89% of the time in the center compared to 8.64% ± 0.91% observed in the control mice (p < 0.001 by t-test). A forced swim test found significantly less depression manifested by immobility in the ANXA2 group. The changes in behavior were accompanied by a significant reduction in serum corticosteroid levels of ANXA2 group compared to controls. Moreover, higher levels of total IgG and p11 expression were found in ANXA2 group brains. Lower levels of circulating anti-ANXA2 Abs were not associated with behavioral changes. CONCLUSIONS: We have established an animal model with high levels of anti-ANXA2 Abs which induced IgG accumulation in the brain and specific anxiolytic and anti-depressive effects. This model promises to further our understanding of autoimmune disease such as APS and to provide better understanding of the role of the ANXA2-p11 complex in the brain.

The Membrane Phospholipid Binding Protein Annexin A2 Promotes Phagocytosis and Nonlytic Exocytosis of Cryptococcus neoformans and Impacts Survival in Fungal Infection.

Cryptococcus neoformans is a fungal pathogen with a unique intracellular pathogenic strategy that includes nonlytic exocytosis, a phenomenon whereby fungal cells are expunged from macrophages without lysing the host cell. The exact mechanism and specific proteins involved in this process have yet to be completely defined. Using murine macrophages deficient in the membrane phospholipid binding protein, annexin A2 (ANXA2), we observed a significant decrease in both phagocytosis of yeast cells and the frequency of nonlytic exocytosis. Cryptococcal cells isolated from Anxa2-deficient (Anxa2(-/-)) bone marrow-derived macrophages and lung parenchyma displayed significantly larger capsules than those isolated from wild-type macrophages and tissues. Concomitantly, we observed significant differences in the amount of reactive oxygen species produced between Anxa2(-/-) and Anxa2(+/+) macrophages. Despite comparable fungal burden, Anxa2(-/-) mice died more rapidly than wild-type mice when infected with C. neoformans, and Anxa2(-/-) mice exhibited enhanced inflammatory responses, suggesting that the reduced survival reflected greater immune-mediated damage. Together, these findings suggest a role for ANXA2 in the control of cryptococcal infection, macrophage function, and fungal morphology.

The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation.

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

Long-term efficacy and downstream mechanism of anti-annexinA2 monoclonal antibody (anti-ANX A2 mAb) in a pre-clinical model of aggressive human breast cancer.

There is considerable direct evidence that calcium binding protein ANX A2 is a potential target for treating aggressive breast cancer. The most compelling data are based on the finding of ANX A2 overexpression in aggressive triple negative human breast cancer (TNBC) cell lines and in human breast cancer tissues. Previously, we and others reported a unique role of ANX A2 in cancer invasion, including breast cancer. Moreover, we demonstrated that anti-ANX A2 mAb-mediated immunoneutralization of ANX A2 inhibited invasive human breast cancer growth in a xenograft model. We further evaluated the long-term effects of multiple treatments with anti-ANX A2 mAb and its mechanism of inhibition on human breast tumor growth. We now demonstrate that three treatments with anti-ANX A2 mAb led to significant inhibition of breast tumor growth in immunodeficient mice, and that the anti-tumor response was demonstrable from day 94. After treatment, we followed tumor growth for 172 days and demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated that anti-ANX A2 mAb treatment caused significant inhibition of conversion of tissue plasminogen activator (tPA) in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathways in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for treatment of aggressive breast cancer.

Annexin A2 Enhances Complement Activation by Inhibiting Factor H.

Factor H is a circulating protein that regulates activation of the alternative pathway (AP) of complement. Mutations and genetic variations of factor H are associated with several AP-mediated diseases, highlighting the critical role of factor H in AP regulation. AP-mediated inflammation is typically triggered by illness or tissue injury, however, and tissue injury can trigger AP activation in individuals with fully functional factor H. This suggests that factor H function is affected by local conditions within tissues. We hypothesized that inducible proteins impair the ability of factor H to locally control the AP, thereby increasing AP activation. We used purified murine factor H to immunoprecipitate binding partners from mouse kidneys. Using immunoaffinity liquid chromatography-mass spectrometry, we identified annexin A2 as a factor H binding partner. Further experiments showed that annexin A2 reduces the binding of factor H to cell surfaces. Recombinant annexin A2 impaired complement regulation by factor H and increased complement activation on renal cell surfaces in vitro and in vivo. In a murine model of acute pneumococcal otitis media, the administration of annexin A2 increased AP-mediated bacterial opsonization and clearance. In conclusion, the local production of annexin A2 within tissues suppresses regulation of the AP by factor H. Annexin A2 can contribute to AP-mediated tissue inflammation by locally impairing factor H function, but it can also improve complement-mediated bacterial clearance.

Annexin A2 binds to endosomes and negatively regulates TLR4-triggered inflammatory responses via the TRAM-TRIF pathway.

Lipopolysaccharide (LPS) derived from Gram-negative bacteria activates plasma membrane signaling via Toll-like receptor 4 (TLR4) on host cells and triggers innate inflammatory responses, but the underlying mechanisms remain to be fully elucidated. Here we reveal a role for annexin A2 (AnxA2) in host defense against infection as anxa2(-/-) mice were highly susceptible to Gram-negative bacteria-induced sepsis with enhanced inflammatory responses. Computing analysis and biochemical experiments identified that constitutive AnxA2 expression facilitated TLR4 internalization and its subsequent translocation into early endosomal membranes. It activated the TRAM-dependent endosomal signaling, leading to the release of anti-inflammatory cytokines. Importantly, AnxA2 deficiency prolonged TLR4-mediated signaling from the plasma membrane, which was attributable to pro-inflammatory cytokine production (IL-6, TNFα and IL-1ß). Thus, AnxA2 directly exerted negative regulation of inflammatory responses through TLR4-initiated TRAM-TRIF pathway occurring on endosomes. This study reveals AnxA2 as a critical regulator in infection-initiated inflammation, which protects the host from excessive inflammatory damage.