SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1-involved EMT in hepatocellular carcinoma.

BACKGROUND: The protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression. METHODS: The modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient's overall survival versus AnxA6 expression level was evaluated by the Kaplan-Meier method. RESULTS: Lys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients. CONCLUSIONS: AnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis.

Annexin A6 mitigates neurological deficit in ischemia/reperfusion injury by promoting synaptic plasticity.

AIMS: Alleviating neurological dysfunction caused by acute ischemic stroke (AIS) remains intractable. Given Annexin A6 (ANXA6)'s potential in promoting axon branching and repairing cell membranes, the study aimed to explore ANXA6's potential in alleviating AIS-induced neurological dysfunction. METHODS: A mouse middle cerebral artery occlusion model was established. Brain and plasma ANXA6 levels were detected at different timepoints post ischemia/reperfusion (I/R). We overexpressed and down-regulated brain ANXA6 and evaluated infarction volume, neurological function, and synaptic plasticity-related proteins post I/R. Plasma ANXA6 levels were measured in patients with AIS and healthy controls, investigating ANXA6 expression's clinical significance. RESULTS: Brain ANXA6 levels initially decreased, gradually returning to normal post I/R; plasma ANXA6 levels showed an opposite trend. ANXA6 overexpression significantly decreased the modified neurological severity score (p = 0.0109) 1 day post I/R and the infarction area at 1 day (p = 0.0008) and 7 day (p = 0.0013) post I/R, and vice versa. ANXA6 positively influenced synaptic plasticity, upregulating synaptophysin (p = 0.006), myelin basic protein (p = 0.010), neuroligin (p = 0.078), and tropomyosin-related kinase B (p = 0.150). Plasma ANXA6 levels were higher in patients with AIS (1.969 [1.228-3.086]) compared to healthy controls (1.249 [0.757-2.226]) (p < 0.001), that served as an independent risk factor for poor AIS outcomes (2.120 [1.563-3.023], p < 0.001). CONCLUSIONS: This study is the first to suggest that ANXA6 enhances synaptic plasticity and protects against transient cerebral ischemia.

SUMOylation of AnxA6 facilitates EGFR-PKCα complex formation to suppress epithelial cancer growth.

BACKGROUND: The Annexin A6 (AnxA6) protein is known to inhibit the epidermal growth factor receptor (EGFR)-extracellular signal regulated kinase (ERK)1/2 signaling upon EGF stimulation. While the biochemical mechanism of AnxA6 inactivating phosphorylation of EGFR and ERK1/2 is not completely explored in cancer cells. METHODS: Cells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1 plasmids to enrich the SUMOylated AnxA6 by immunoprecipitation, and the modification level of AnxA6 by SUMO1 was detected by Western blot against SUMO1 antibody. The SUMOylation level of AnxA6 was compared in response to chemical SUMOylation inhibitor treatment. AnxA6 SUMOylation sites were further identified by LC-MS/MS and amino acid site mutation validation. AnxA6 gene was silenced through AnxA6 targeting shRNA-containing pLKO.1 lentiviral transfection in HeLa cells, while AnxA6 gene was over-expressed within the Lenti-Vector carrying AnxA6 or mutant AnxA6K299R plasmid in A431 cells using lentiviral infections. Moreover, the mutant plasmid pGFP-EGFRT790M/L858R was constructed to test AnxA6 regulation on EGFR mutation-induced signal transduction. Moreover, cell proliferation, migration, and gefitinib chemotherapy sensitivity were evaluated in HeLa and A431 cells under AnxA6 konckdown or AnxA6 overexpression by CCK8, colony form and wound healing assays. And tumorigenicity in vivo was measured in epithelial cancer cells-xenografted nude mouse model. RESULTS: AnxA6 was obviously modified by SUMO1 conjugation within Lys (K) residues, and the K299 was one key SUMOylation site of AnxA6 in epithelial cancer cells. Compared to the wild type AnxA6, AnxA6 knockdown and its SUMO site mutant AnxA6K299R showed less suppression of dephosphorylation of EGFR-ERK1/2 under EGF stimulation. The SUMOylated AnxA6 was prone to bind EGFR in response to EGF inducement, which facilitated EGFR-PKCα complex formation to decrease the EGF-induced phosphorylation of EGFR-ERK1/2 and cyclin D1 expression. Similarly, AnxA6 SUMOylation inhibited dephosphorylation of the mutant EGFR, thereby impeding EGFR mutation-involved signal transduction. Moreover, AnxA6 knockdown or the K299 mutant AnxA6K299R conferred AnxA6 inability to suppress tumor progression, resulting in drug resistance to gefitinib in epithelial cancer cells. And in epithelial cancer cells-xenografted nude mouse model, both the weight and size of tumors derived from AnxA6 knockdown or AnxA6K299R mutation-expressing cells were much greater than that of AnxA6-expressing cells. CONCLUSIONS: Besides EGFR gene mutation, protein SUMOylation modification of EGFR-binding protein AnxA6 also functions pivotal roles in mediating epithelial cancer cell growth and gefitinib drug effect. Video Abstract.

Annexin A6 mediates calcium-dependent exosome secretion during plasma membrane repair.

Exosomes are an extracellular vesicle (EV) subtype that is secreted upon the fusion of multivesicular bodies (MVBs) with the plasma membrane. Exosomes may participate in intercellular communication and have utility as disease biomarkers; however, little is known regarding the physiological stimuli that induce their secretion. Ca2+ influx promotes exosome secretion, raising the possibility that exosomes are secreted during the Ca2+-dependent plasma membrane repair of tissues damaged by mechanical stress in vivo. To determine whether exosomes are secreted upon plasma membrane damage, we developed sensitive assays to measure exosome secretion in intact and permeabilized cells. Our results suggest that exosome secretion is coupled to Ca2+-dependent plasma membrane repair. We find that annexin A6 (ANXA6), a well-known plasma membrane repair protein, is recruited to MVBs in the presence of Ca2+ and required for Ca2+-dependent exosome secretion, both in intact and in permeabilized cells. ANXA6 depletion stalls MVBs at the cell periphery, and ANXA6 truncations localize to different membranes, suggesting that ANXA6 may serve to tether MVBs to the plasma membrane. We find that cells secrete exosomes and other EVs upon plasma membrane damage and propose that repair-induced secretion may contribute to the pool of EVs present within biological fluids.

Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

Hypoxia-Inducible Expression of Annexin A6 Enhances the Resistance of Triple-Negative Breast Cancer Cells to EGFR and AR Antagonists.

Physiological changes such as hypoxia in the tumor microenvironment (TME) endow cancer cells with malignant properties, leading to tumor recurrence and rapid progression. Here, we assessed the effect of hypoxia (1% Oxygen) on the tumor suppressor Annexin A6 (AnxA6) and the response of triple-negative breast cancer (TNBC) cells to epidermal growth factor receptor (EGFR) and androgen receptor (AR) targeted therapies. We demonstrate that brief exposure of TNBC cells to hypoxia (within 24 h) is associated with down regulation of AnxA6 while > 24 h exposure cell type dependently stimulated the expression of AnxA6. Hypoxia depicted by the expression and stability of HIF-1/2α led to up regulation of the HIF target genes SLC2A1, PGK1 as well as AR and the AR target genes FABP-4 and PPAR-γ, but the cellular levels of AnxA6 protein decreased under prolonged hypoxia. Down regulation of AnxA6 in TNBC cells inhibited, while AnxA6 over expression enhanced the expression and cellular levels of HIF-1/2α, SLC2A1 and PGK1. RNAi mediated inhibition of hypoxia induced AnxA6 expression also strongly inhibited glucose uptake and ROS production in AnxA6 expressing TNBC cells. Using a luciferase reporter assay, we confirm that short-term exposure of cells to hypoxia inhibits while prolonged exposure of cells to hypoxia enhances AnxA6 promoter activity in HEK293T cells. Compared to cells cultured under normoxia, TNBC cells were more resistant to lapatinib under hypoxic conditions, and the downregulation of AnxA6 sensitized the cells to EGFR as well as AR antagonists. These data suggest that AnxA6 is a hypoxia inducible gene and that targeting AnxA6 upregulation may be beneficial in overcoming TNBC resistance to EGFR and/or AR targeted therapies.

Mineralization Profile of Annexin A6-Harbouring Proteoliposomes: Shedding Light on the Role of Annexin A6 on Matrix Vesicle-Mediated Mineralization.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles' nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS-CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS-CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles' nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS-CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.

A conserved annexin A6-mediated membrane repair mechanism in muscle, heart, and nerve.

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 µM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.

The mouse Anxa6/miR-9-5p/Anxa2 axis modulates TGF-ß1-induced mouse hepatic stellate cell (mHSC) activation and CCl4-caused liver fibrosis.

Chronic liver disease such as hepatic fibrosis is a major cause of morbidity and mortality and has been related to high individual risk of hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) activation is a central event of hepatic fibrosis progression. In this study, the up-regulation of lncRNA ANXA2P2 (mouse Anxa6) was found in liver fibrosis. Within CCl4-caused liver fibrosis murine model, Anxa6 knockdown partially ameliorated CCl4-induced hepatic fibrosis and blocked the PI3K/Akt signaling activation. In TGF-ß1-stimulated HSCs, Anxa6 knockdown partially inhibited TGF-ß1-induced HSC activation and blocked the PI3K/Akt signaling activation. Mouse Anxa6 downstream mmu-miR-9-5p directly targeted Anxa2; Anxa6 negatively regulated mmu-miR-9-5p, and mmu-miR-9-5p negatively regulated mouse Anxa2. In TGF-ß1-stimulated HSCs, miR-9-5p inhibitor promoted TGF-ß1-induced HSC activation and PI3K/Akt signaling activation, whereas Anxa2 knockdown exerted opposite effects; Anxa2 knockdown significantly attenuated miR-9-5p inhibitor effects upon TGF-ß1-stimulated HSCs. In conclusion, lncRNA ANXA2P2 (mouse Anxa6) expression is up-regulated in hepatic fibrosis and exerts pro-fibrotic effects on CCl4-caused liver fibrosis model mice and TGF-ß1-stimulated HSCs. The mouse Anxa6/miR-9-5p/Anxa2 axis and the PI3K/Akt pathway might participate in the functions of lncRNA ANXA2P2 (mouse Anxa6) on hepatic fibrosis.

Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions.

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

CRISPR-Cas9 mediated knockout of AnxA6 gene enhances influenza A virus replication in low-permissive HEK293FT cell line.

Using cell cultures of human origin for the propagation of influenza virus is an attractive way to preserve its glycosylation profile and antigenic properties, which is essential in influenza surveillance and vaccine production. However, only few cell lines are highly permissive to influenza virus, and none of them are of human origin. The barrier might be associated with host restriction factors inhibiting influenza growth, such as AnxA6 protein counteracting the process of influenza virion packaging. In the presented work we explore the CRISPR-Cas9 mediated knockout of ANXA6 gene as a way to overcome the host restriction barrier and increase the susceptibility of human cell line to influenza infection. By CRISPR-Cas9 genome editing we modified HEK293FT cells and obtained several clones defective in the ANXA6 gene. The replication of the influenza A virus in original HEK293FT cells and the HEK293FT-ANXA6-/- mutant cells was compared in growth curve experiments. By combination of methods including TCID assay and flow cytometry we showed that accumulation of influenza A virus in the mutant HEK293FT-ANXA6-/- cells significantly exceeded the virus titer in the original HEK293FT cells.

Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer.

Exosomes are carriers of intercellular information that regulate the tumor microenvironment, and they have an essential role in drug resistance through various mechanisms such as transporting RNA molecules and proteins. Nevertheless, their effects on gemcitabine resistance in triple-negative breast cancer (TNBC) are unclear. In the present study, we examined the effects of exosomes on TNBC cell viability, colony formation, apoptosis, and annexin A6 (ANXA6)/EGFR expression. We addressed their roles in gemcitabine resistance and the underlying mechanism. Our results revealed that exosomes derived from resistant cancer cells improved cell viability and colony formation and inhibited apoptosis in sensitive cancer cells. The underlying mechanism included the transfer of exosomal ANXA6 from resistant cancer cells to sensitive cancer cells. Isobaric peptide labeling-liquid chromatography-tandem mass spectrometry and western blotting revealed that ANXA6 was upregulated in resistant cancer cells and their derived exosomes. Sensitive cancer cells exhibited resistance with increased viability and colony formation and decreased apoptosis when ANXA6 was stably overexpressed. On the contrary, knockdown ANXA6 restored the sensitivity of cells to gemcitabine. Co-immunoprecipitation expression and GST pulldown assay demonstrated that exosomal ANXA6 and EGFR could interact with each other and exosomal ANXA6 was associated with the suppression of EGFR ubiquitination and downregulation. While adding lapatinib reversed gemcitabine resistance induced by exosomal ANXA6. Moreover, ANXA6 and EGFR protein expression was correlated in TNBC tissues, and exosomal ANXA6 levels at baseline were lower in patients with highly sensitive TNBC than those with resistant TNBC when treated with first-line gemcitabine-based chemotherapy. In conclusion, resistant cancer cell-derived exosomes induced gemcitabine resistance via exosomal ANXA6, which was associated with the inhibition of EGFR ubiquitination and degradation. Exosomal ANXA6 levels in the serum of patients with TNBC might be predictive of the response to gemcitabine-based chemotherapy.

Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells.

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate Pi and Ca2+ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PPi), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.

Extracellular Vesicles from Cancer-Associated Fibroblasts Containing Annexin A6 Induces FAK-YAP Activation by Stabilizing ß1 Integrin, Enhancing Drug Resistance.

Extracellular vesicles (EV) from cancer-associated fibroblasts (CAF) are composed of diverse payloads. Although CAFs impact the aggressive characteristics of gastric cancer cells, the contribution of CAF-EV to gastric cancer progression has not been elucidated. Here, we investigated the molecular mechanism of the changes in gastric cancer characteristics induced by CAF-EV. CAF abundance in gastric cancer tissues was associated with poor prognosis of patients with gastric cancer receiving chemotherapy. Moreover, CAF-EV induced tubular network formation and drug resistance of gastric cancer cells in the extracellular matrix (ECM). Comprehensive proteomic analysis of CAF-EV identified that Annexin A6 plays a pivotal role in network formation and drug resistance of gastric cancer cells in the ECM via activation of ß1 integrin-focal adhesion kinase (FAK)-YAP. A peritoneal metastasis mouse model revealed that CAF-EV induced drug resistance in peritoneal tumors, and inhibition of FAK or YAP efficiently attenuated gastric cancer drug resistance in vitro and in vivo. These findings demonstrate that drug resistance is conferred by Annexin A6 in CAF-EV and provide a potential avenue for overcoming gastric cancer drug resistance through the inhibition of FAK-YAP signaling in combination with conventional chemotherapeutics. SIGNIFICANCE: This study elucidates a novel molecular mechanism through which Annexin A6 in CAF-EV activates FAK-YAP by stabilizing ß1 integrin at the cell surface of gastric cancer cells and subsequently induces drug resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/16/3222/F1.large.jpg.

Annexin-A6 in Membrane Repair of Human Skeletal Muscle Cell: A Role in the Cap Subdomain.

Defects in membrane repair contribute to the development of some muscular dystrophies, highlighting the importance to decipher the membrane repair mechanisms in human skeletal muscle. In murine myofibers, the formation of a cap subdomain composed notably by annexins (Anx) is critical for membrane repair. We applied membrane damage by laser ablation to human skeletal muscle cells and assessed the behavior of annexin-A6 (AnxA6) tagged with GFP by correlative light and electron microscopy (CLEM). We show that AnxA6 was recruited to the site of membrane injury within a few seconds after membrane injury. In addition, we show that the deficiency in AnxA6 compromises human sarcolemma repair, demonstrating the crucial role played by AnxA6 in this process. An AnxA6-containing cap-subdomain was formed in damaged human myotubes in about one minute. Through transmission electron microscopy (TEM), we observed that extension of the sarcolemma occurred during membrane resealing, which participated in forming a dense lipid structure in order to plug the hole. By properties of membrane folding and curvature, AnxA6 helped in the formation of this tight structure. The compaction of intracellular membranes-which are used for membrane resealing and engulfed in extensions of the sarcolemma-may also facilitate elimination of the excess of lipid and protein material once cell membrane has been repaired. These data reinforce the role played by AnxA6 and the cap subdomain in membrane repair of skeletal muscle cells.

Selective Degradation Permits a Feedback Loop Controlling Annexin A6 and Cholesterol Levels in Endolysosomes of NPC1 Mutant Cells.

We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann-Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.

Reciprocal expression of Annexin A6 and RasGRF2 discriminates rapidly growing from invasive triple negative breast cancer subsets.

Actively growing tumors are often histologically associated with Ki67 positivity, while the detection of invasiveness relies on non-quantitative pathologic evaluation of mostly advanced tumors. We recently reported that reduced expression of the Ca2+-dependent membrane-binding annexin A6 (AnxA6) is associated with increased expression of the Ca2+ activated RasGRF2 (GRF2), and that the expression status of these proteins inversely influence the growth and motility of triple negative breast cancer (TNBC) cells. Here, we establish that the reciprocal expression of AnxA6 and GRF2 is at least in part, dependent on inhibition of non-selective Ca2+ channels in AnxA6-low but not AnxA6-high TNBC cells. Immunohistochemical staining of breast cancer tissues revealed that compared to non-TNBC tumors, TNBC tumors express lower levels of AnxA6 and higher Ki67 expression. GRF2 expression levels strongly correlated with high Ki67 in pretreatment biopsies from patients with residual disease and with residual tumor size following chemotherapy. Elevated AnxA6 expression more reliably identified patients who responded to chemotherapy, while low AnxA6 levels were significantly associated with shorter distant relapse-free survival. Finally, the reciprocal expression of AnxA6 and GRF2 can delineate GRF2-low/AnxA6-high invasive from GRF2-high/AnxA6-low rapidly growing TNBCs. These data suggest that AnxA6 may be a reliable biomarker for distant relapse-free survival and response of TNBC patients to chemotherapy, and that the reciprocal expression of AnxA6 and GRF2 can reliably delineate TNBCs into rapidly growing and invasive subsets which may be more relevant for subset-specific therapeutic interventions.

Annexin A6 Is Critical to Maintain Glucose Homeostasis and Survival During Liver Regeneration in Mice.

BACKGROUND AND AIMS: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. APPROACH AND RESULTS: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. CONCLUSIONS: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.

Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization.

Annexin A6 (AnxA6) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs are a special class of extracellular vesicles that serve as a nucleation site during cartilage, bone, and mantle dentin mineralization. In this study, we assessed the localization of AnxA6 in the MV membrane bilayer using native MVs and MV biomimetics. Biochemical analyses revealed that AnxA6 in MVs can be divided into three distinct groups. The first group corresponds to Ca2+-bound AnxA6 interacting with the inner leaflet of the MV membrane. The second group corresponds to AnxA6 localized on the surface of the outer leaflet. The third group corresponds to AnxA6 inserted in the membrane's hydrophobic bilayer and co-localized with cholesterol (Chol). Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV membrane bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, with and without Ca2+, we confirmed that, in agreement with the biochemical data, AnxA6 interacted differently with the MV membrane. Thermodynamic analyses based on the measurement of surface pressure exclusion (πexc), enthalpy (ΔH), and phase transition cooperativity (Δt1/2) showed that AnxA6 interacted with DPPC and 9:1 DPPC:DPPS systems and that this interaction increased in the presence of Chol. The selective recruitment of AnxA6 by Chol was observed in MVs as probed by the addition of methyl-ß-cyclodextrin (MßCD). AnxA6-lipid interaction was also Ca2+-dependent, as evidenced by the increase in πexc in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in ΔH in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in the presence of Ca2+ compared to DPPC zwitterionic bilayers. The interaction of AnxA6 with DPPC and 9:1 DPPC:DPPS systems was distinct even in the absence of Ca2+ as observed by the larger change in Δt1/2 in 9:1 DPPC:DPPS vesicles as compared to DPPC vesicles. Protrusions on the surface of DPPC proteoliposomes observed by atomic force microscopy suggested that oligomeric AnxA6 interacted with the vesicle membrane. Further work is needed to delineate possible functions of AnxA6 at its different localizations and ways of interaction with lipids.