Abstract B26: Role of exosomal annexin a2 in angiogenesis and breast cancer metastasis

Abstract

Abstract Background: Early detection of cancer using circulating biomarkers is a realistic possibility with the discovery of exosomes. Cells under both physiological and pathological conditions secrete a wide array of membranous vesicles containing specific protein and RNA signatures. Exosomes, which are 40-100 nm in size, comprise a major portion of these vesicles. Exosomes play important roles in promoting tumor progression and metastasis, but the mechanisms by which they act are not yet understood. Annexin A2 (AnxA2) is a 36 kDa calcium dependent phospholipid-binding protein up-regulated in many cancer types and promotes tumorigenesis and angiogenesis by acting as a receptor for angiostatin and tissue plasminogen activator (t-PA) leading to plasmin mediated angiogenesis and metastasis. Recently, it was found that AnxA2 is one of the most commonly expressed proteins in exosomes, but its function has never been characterized. We propose to explore the correlation and functionality of exosome associated AnxA2 (exo-AnxA2) in breast cancer. Methods: We used two breast cancer models: MCF10 progression model (MCF10A non to correlate exo-AnxA2 with breast cancer progression, and MDA-MB-231/ its organ specific metastatic variants (BoM-1833 bone, MDA-MB-231-831 brain and MDA-MB-231-4175 lungs). Exosome characterization was performed via Western blotting (WB), particle size analyzer, Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). In vitro and in vivo angiogenesis assays were performed to explore the role of exo-AnxA2 in angiogenesis. AnxA2 targeting peptide (LCKLSL) was used to inhibit exo-AnxA2. For metastasis studies, experimentally induced (intracardiac) and spontaneous (mammary fat pad) metastasis models were used. Animals were primed for one month (lateral tail vein injection) with PBS or MDA-MB-231/variants derived exosomes having WT AnxA2 (exo-WTAnxA2) or sh-AnxA2 (exo-shAnxA2). After one month of priming, the animals were challenged with luciferase positive MDA-MB-231/variants and the differences in the extent of metastasis were studied. We performed clinical analysis AnxA2 levels in 50 breast cancer serum samples and 50 age matched normal serum samples by analyzing total serum AnxA2 levels and serum exosomal AnxA2 levels via ELISA. We also correlated the AnxA2 levels with types of cancer (ER, PR, HER2 or Triple negative). Further, we correlated the exosomal AnxA2 levels with stage (early versus late stage) and metastatic sites (no metastasis, lung, brain and/or bone) of patients. Result: AFM and WB characterization of exo-AnxA2 along the progression model revealed that AnxA2 is highly expressed in cancer exosomes vs. non-malignant and pre-malignant exosomes. Both in vitro and in vivo angiogenesis studies with LCKLSL showed exo-AnxA2 to be a potent inducer of angiogenesis (∼ 45% and ∼42.4% decrease with LCKLSL respectively). Furthermore, both the metastasis models showed a significant decrease in the extent of organ specific metastasis (lungs ∼43%, brain ∼ 30% and bone ∼41%) in animals primed with exo-shAnxA2 vs. exo-WTAnxA2. Histological analyses of the organs showed increased micro and macro metastasis in the brain, lungs and bones in exo-WTAnxA2 primed animals when compared to exo-shAnxA2. Clinical analysis of the serum samples revealed that although total serum AnxA2 is increased in breast cancer serum samples vs. normal, the correlation was much better between high exo-AnxA2 levels and metastatic breast cancer. Conclusion: Exo-AnxA2 plays a major role in promoting angiogenesis and breast cancer metastasis both in vitro and in vivo. Further, exo-AnxA2 is over expressed in breast cancer serum samples vs. control. Thus, exo-AnxA2 can be a potential diagnostic and prognostic marker in breast cancer patients to detect and monitor metastasis. All the authors accept the responsibility for the statements made in the abstract. (Supported by NIMHHD grant under Award Number P20MD006882) Citation Format: Sayantan Maji, Ann Marilyn Leitch, Irina Akopova, Phung Nguyen, Jamboor K. Vishwanatha. Role of exosomal annexin a2 in angiogenesis and breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B26.