A novel inhibitory anti-invasive MAb isolated using phenotypic screening highlights AnxA6 as a functionally relevant target protein in pancreatic cancer.

BACKGROUND: Discovery and validation of new antibody tractable targets is critical for the development of new antibody therapeutics to address unmet needs in oncology. METHODS: A highly invasive clonal variant of the MDA-MB-435S cell line was used to generate monoclonal antibodies (MAbs), which were screened for anti-invasive activity against aggressive cancer cells in vitro. The molecular target of selected inhibitory MAb 9E1 was identified using immunoprecipitation/liquid chromatography-tandem mass spectrometry. The potential anti-tumour effects of MAb 9E1 were investigated in vitro together with immunohistochemical analysis of the 9E1 target antigen in normal and cancer tissues. RESULTS: MAb 9E1 significantly decreases invasion in pancreatic, lung squamous and breast cancer cells and silencing of its target antigen, which was revealed as AnxA6, leads to markedly reduced invasive capacity of pancreatic and lung squamous cancer in vitro. IHC using MAb 9E1 revealed that AnxA6 exhibits a high prevalence of membrane immunoreactivity across aggressive tumour types with restricted expression observed in the majority of normal tissues. In pancreatic ductal adenocarcinoma, high AnxA6 IHC score correlated with the presence of tumour budding at the invasive front of tumours (P=0.082), the presence of perineural invasion (P= <0.0001) and showed a weak correlation with reduced survival (P=0.2242). CONCLUSIONS: This study highlights the use of phenotypic hybridoma screening as an effective strategy to select a novel function-blocking MAb, 9E1 with anti-cancer activity in vitro. Moreover, through characterisation of the 9E1 target antigen, AnxA6, our findings support further investigation of AnxA6 as a potential candidate target for antibody-mediated inhibition of pancreatic cancer.

Immunological development and cardiovascular function are normal in annexin VI null mutant mice.

Annexins are calcium-binding proteins of unknown function but which are implicated in important cellular processes, including anticoagulation, ion flux regulation, calcium homeostasis, and endocytosis. To gain insight into the function of annexin VI, we performed targeted disruption of its gene in mice. Matings between heterozygous mice produced offspring with a normal Mendelian pattern of inheritance, indicating that the loss of annexin VI did not interfere with viability in utero. Mice lacking annexin VI reached sexual maturity at the same age as their normal littermates, and both males and females were fertile. Because of interest in the role of annexin VI in cardiovascular function, we examined heart rate and blood pressure in knockout and wild-type mice and found these to be identical in the two groups. Similarly, the cardiovascular responses of both sets of mice to septic shock were indistinguishable. We also examined components of the immune system and found no differences in thymic, splenic, or bone marrow lymphocyte levels between knockout and wild-type mice. This is the first study of annexin knockout mice, and the lack of a clear phenotype has broad implications for current views of annexin function.

The identification and differential expression of calcium-binding proteins associated with ocular melanoma.

Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

Annexin 6 immunoreactivity in select cell populations in the rat brain.

We examined the distribution of annexin 6 (ANX6) in rat brain with immunohistochemistry (IHC). Several neuronal cell populations were intensely labeled with the ANX6 monoclonal antibody (MAb), including layer 5 of neocortex, the lateral septum, the lateral hypothalamic area, the red nucleus, and the Purkinje cell layer in cerebellum. Neuronal immunolabeling was localized to the nucleus and the cytosol. Darkly stained ANX6-immunoreactive glia, with the morphology characteristic of astrocytes, were abundant in the hippocampus, substantia nigra reticulata, and cerebellum. Evidence suggests that ANX6 may function in neuronal and glial calcium-dependent processes.

Smooth muscle actomyosin promotes Ca2+-dependent interactions between annexin VI and detergent-insoluble glycosphingolipid-enriched membrane domains.

The mechanical link coupling cytoskeletal and contractile proteins to the sarcolemma of smooth muscle cells is essential for transmitting tension from the cell's interior to exterior. In addition to the well-characterized actin-integrin associations present in adhaerens junctions, our recent work has postulated the existence of a reversible annexin-dependent membrane-cytoskeleton complex, forged in response to a rise in intracellular Ca2+ concentration following smooth muscle cell stimulation (Babiychuk et al., J. Biol Chem. 1999, 274, 35191-35195). Detailed biochemical characterization of the interactions responsible for the formation of this complex revealed that annexins II and VI interact with actomyosin, or detergent-insoluble glycosphingolipid-enriched membrane domains (rafts) purified from smooth muscle, in a concentration- and Ca2+-dependent manner. Annexin II interacted with lipid rafts with high Ca2+-sensitivity, while for annexin VI this interaction required non-physiologically high concentrations of free Ca2+. However, the Ca2+-sensitivity of the latter interaction strongly increased in the presence of purified smooth muscle actomyosin. The detailed biochemical analysis of the interactions occurring between annexin II, annexin VI, actomyosin and rafts suggests that annexins regulate sarcolemmal organization during smooth muscle cell contraction.

A case of neonatal lupus erythematosus presenting delayed dilated cardiomyopathy with circulating autoantibody to annexin A6.

Patients with neonatal lupus erythematosus (NLE) often have congenital heart block with or without heart failure and are born to mothers who have anti-SS-A and/or anti-SS-B antibodies. NLE has been considered to result from the placental transmission of maternal autoantibodies into the fetal circulation causing myocardial damage. We report a case of NLE with congenital heart block who had undergone pacemaker implantation at the age of 17, and then developed dilated cardiomyopathy (DCM) at the age of 19, which is much later than in most other cases. The patient's mother was positive for anti-SS-A and anti-SS-B antibodies, whereas the patient was negative for both anti-SS-A and anti-SS-B antibodies. There were some autoantibodies against cell surface antigens of cardiac myocytes in the serum from the patient, and annexin A6 was identified as one of the autoantigens. This is the first report demonstrating that annexin A6 is involved in the myocardial injury in patients with NLE. The results indicate that inhibition of annexin A6 function may prevent autoantibody-mediated myocardial injury in at least some cases of DCM.

High levels of antibodies to annexins V and VI in patients with rheumatoid arthritis.

OBJECTIVE: Glucocorticoids are powerful antiinflammatory agents widely used for the treatment of rheumatoid arthritis (RA). Synthesis and/or secretion of annexin I (A-I) is induced by these steroids. Annexins V and VI are also found extracellularly but are not induced by glucocorticoids. Annexins may be potent antagonists of phospholipase A2 (PLA2). Since autoantibodies to A-I have been reported in patients with RA, we studied the reactivity of sera from patients with RA to A-V and A-VI. METHODS: Sera from 26 patients with RA were assessed for anti-A-V and anti-A-VI antibodies and compared with sera from 26 sex/age matched healthy subjects. IgG and IgM antibodies were analyzed in an ELISA: A correlation study with disease activity and corticosteroid treatment schedule was performed. RESULTS: Sera from patients with RA contained significantly higher levels of IgG [anti-A-V and anti-A-VI] autoantibodies than control sera, both being correlated. This rise in antiannexin antibody titers was correlated with the RA activity score, and negatively correlated with the daily dose of corticosteroids. CONCLUSION: High levels of IgG (anti-A-V and anti-A-VI) antibodies were found in sera from patients with RA. We suggest that antiannexin autoantibodies may play a role in the clinical course of RA by impairing the anti-PLA2 effect of annexins.