Knockdown of annexin A5 restores gefitinib sensitivity by promoting G2/M cell cycle arrest.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, including gefitinib, are first-line drugs against advanced non-small cell lung cancer with activating EGFR mutations. However, the development of resistance to such drugs is a major clinical challenge. METHODS: The role of annexin A5 in resistance to EGFR tyrosine kinase inhibitors was investigated by qPCR and western blot of relevant molecules, by CCK8 and EdU assay of cell proliferation and viability, by annexin V/propidium iodide assay of apoptosis and cell cycle distribution, by JC-1 assay of mitochondrial integrity, and by xenograft assay of tumorigenicity. RESULTS: We found that annexin A5 is upregulated in gefitinib-resistant cell lines, as well as in clinical specimens resistant to EGFR tyrosine kinase inhibitors. Accordingly, knockdown of the gene from gefitinib-resistant cells restores gefitinib sensitivity in vitro and in vivo by downregulating polo-like kinase 1 signal pathway, thereby inducing mitochondrial damage, caspase activation, cell cycle arrest at G2/M, and, finally, apoptosis. CONCLUSIONS: The data indicate that annexin A5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and G2/M cell cycle arrest, and is thus a potential therapeutic target in non-small cell lung cancers resistant to EGFR tyrosine kinase inhibitors.

Annexin A5 Involvement in Bone Overgrowth at the Enthesis.

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5-/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5-/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5-/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5-/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5-/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5-/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle-mediated mineralization, but lack of collagen X affects hematopoiesis and the Th1/Th2 response.

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle-mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X-deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) newborns, whereas endochondral ossification and mineralization were not affected in 13-day- and 1-month-old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1-month- and 1-year-old mutants even though the diaphyseal circumference was reduced in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) mice. The percentage of naive immature IgM(+) /IgM(+) B cells and peripheral T-helper cells were increased in Col10a1(-/-) and AnxA5(-/-) AnxA6(-/-) Col10a1(-/-) mutants, and activated splenic T cells isolated from Col10a1(-/-) mice secreted elevated levels of IL-4 and GM-CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process.

Annexin A5 stimulates autophagy and inhibits endocytosis.

Macroautophagy is a major lysosomal catabolic process activated particularly under starvation in eukaryotic cells. A new organelle, the autophagosome, engulfs cytoplasmic substrates, which are degraded after fusion with endosomes and/or lysosomes. During a shotgun proteome analysis of purified lysosomal membranes from mouse fibroblasts, a Ca(2+)-dependent phospholipid-binding protein, annexin A5, was found to increase on lysosomal membranes under starvation. This suggests a role for this protein, an abundant annexin with a still unknown intracellular function, in starvation-induced lysosomal degradation. Transient overexpression and silencing experiments showed that annexin A5 increased lysosomal protein degradation, and colocalisation experiments, based on GFP sensitivity to lysosomal acidic pH, indicated that this was mainly the result of inducing autophagosome-lysosome fusion. Annexin A5 also inhibited the endocytosis of a fluid-phase marker and cholera toxin, but not receptor-mediated endocytosis. Therefore, we propose a double and opposite role of annexin A5 in regulating the endocytic and autophagic pathways and the fusion of autophagosomes with lysosomes and endosomes.

Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization.

Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo.

AnnexinA5 renders dead tumor cells immunogenic--implications for multimodal cancer therapies.

Multimodal tumor therapies should aim not only to kill the tumor cells, but also to stimulate a specific immune response to keep residual tumor (stem) cells and metastases under control. Apoptotic cells are mostly noninflammatory or even anti-inflammatory while necrotic cells stimulate the immune system. Whether the immunogenicity of apoptotic tumor cells can be increased by interfering with their swift and phosphatidylserine (PS)-dependent clearance by macrophages was examined. AnnexinA5 (AnxA5) is a naturally occurring highly specific ligand for PS. Proteins of the annexin family are characterized by a selective affinity for phospholipids in the presence of Ca2+ ions. The phagocytosis by macrophages of irradiated, apoptotic tumor cells (ITC) was partially inhibited when the ITC were preincubated with AnxA5. Activated macrophages secreted higher amounts of TNFalpha and IL-1beta after contact with ITC plus AnxA5 in comparison with ITC alone, while the amount of TGF-beta was decreased. Macrophages of AnxA5-deficient mice showed an increased phagocytosis of dead cells. Wild-type mice, where endogenous AnxA5 is present, displayed a significantly faster decline in size of allogeneic tumors in comparison with AnxA5-deficient animals. The addition of AnxA5 to ITC vaccines increased the percentage of tumor-free mice in syngeneic tumor protection and tumor cure assays. AnxA5 alone led to a retard of syngeneic tumor growth that was, however, less pronounced in comparison to treatment of the tumor with ionizing irradiation. In conclusion, AnxA5 disturbs the PS-dependent clearance by macrophages of dying tumor cells, leading to the accumulation of the latter, to the occurrence of secondary necrotic cells, and to an increased uptake of the dead cells by dendritic cells. Tumor cure appendages with dead tumor cells should be performed with AnxA5 as an immune stimulator and could be combined with irradiation, chemotherapy, and hyperthermia to induce immunogenic cell death forms in vivo or ex vivo.

The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti-inflammatory phenotype.

Proteins of the annexin family bind to phospholipids in a Ca(2+) dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.

Expression and thyroid hormone regulation of annexins in the anterior pituitary.

Due to their property to bind to phospholipids in a Ca(2)(+)-dependent manner, proteins of the annexin superfamily are involved in many membrane-related events and thus in various forms of physiological and pathological processes. We were therefore interested in analyzing the mRNA expression of the annexins in the severely disorganized pituitaries of the athyroid Pax8(-/-) mice in comparison with that of control animals. In neither condition was mRNA expression of the annexins A3, A7, A8, A9, A11, and A13 detectable. The annexins A2, A4, and A6 were equally expressed in wild-type and Pax8(-/-) mice. Transcript levels of A1 and A10 were highly increased and those of A5 were significantly decreased in the athyroid mutants compared with controls. Treatment of Pax8(-/-) mice with physiological doses of thyroxine for 3 days normalized the mRNA expression of A1, A5, and A10 indicating that the expression of these annexins is directly regulated by thyroid hormone (TH). Since A5 exhibits by far the highest transcript levels of all annexins in the pituitary and its regulation by TH could be also confirmed at the protein level, we analyzed the mRNA expression of pituitary hormones in A5(-/-) mice. In these mutants, only the beta-FSH mRNA expression was found to be significantly reduced, while the mRNA expression levels of the other pituitary hormones were not altered. These results support the concept that annexins might serve important albeit redundant functions as modulators of pituitary hormone secretion.

DT40 cells lacking the Ca2+-binding protein annexin 5 are resistant to Ca2+-dependent apoptosis.

Annexins are widely expressed Ca(2+)-dependent phospholipid-binding proteins with poorly understood physiological roles. Proposed functions include Ca(2+) channel activity and vesicle trafficking, but neither have been proven in vivo. Here we used targeted gene disruption to generate B-lymphocytes lacking annexin 5 (Anx5) expression and show that this results in reduced susceptibility to a range of apoptotic stimuli. By comparison B-lymphocytes lacking annexin 2 (Anx2) showed no such resistance, providing evidence that this effect is specific to loss of Anx5. The defect in the ANX5(-/-) cells occurs early in the apoptotic program before nuclear condensation, caspase 3 activation, and cell shrinkage, but downstream of an initial Ca(2+) influx. Only UVA/B irradiation induced similar levels of apoptosis in wild-type and ANX5(-/-) cells. Unexpectedly, ANX5(-/-) cells permeabilized in vitro also failed to release mitochondrial cytochrome C, suggesting a possible mechanism for their resistance to apoptosis. These findings demonstrate a role for Anx5 in determining the susceptibility of B-lymphocytes to apoptosis.

The annexinopathies: a new category of diseases.

The annexins are a family of highly homologous phospholipid binding proteins, which share a four-domain structure, with one member of the family - annexin VI - having a duplication consisting of eight domains. Thus far, ten annexins have been described in mammals. Although the biological functions of the annexins have not been definitively established, two human diseases involving annexin abnormalities ('annexinopathies') have been identified as of the time of writing. Overexpression of annexin II occurs in the leukocytes of a subset of patients having a hemorrhagic form of acute promyelocytic leukemia. Underexpression of annexin V occurs on placental trophoblasts in the antiphospholipid syndrome and in preeclampsia. Also, an animal model has been described in which annexin VII is underexpressed and is associated with disease, but the relevance of this animal model to human disease is not yet understood. Future research is likely to elucidate additional 'annexinopathies'.