Exosomal AnnexinA2 promotes angiogenesis and breast cancer metastasis

Purpose: 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 40-100nm in size, comprise a major portion of these vesicles. Annexin A2 (AnxA2) is a 36 KD Ca+2 dependent phospholipid-binding protein up-regulated in many cancer types and implicated in promoting tumorigenesis and angiogenesis. Cancer cells have been shown to secrete more AnxA2 and it is also highly expressed in the exosomes; but the function of exosomal AnxA2 (exo-AnxA2) has never been studied. We hypothesize that exo-AnxA2 promotes angiogenesis and breast cancer metastasis and we propose to explore this in our study. Methods: Exosomes were isolated from MCF10A progression model for progression studies. For the metastasis studies MDA-MB-231 and its organ specific metastatic variants MDA-MB-831 (brain met) and MDA-MB-4175 (lung met) were used. They were characterized via Western blotting, particle size analyzer, transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). In vitro and in vivo angiogenesis assays were performed to study the role of exo-AnxA2 in angiogenesis. Exo-AnxA2 was either inhibited by use of LCKLSL inhibitory peptide or knocked down in these studies. Tail vein and intracardiac injection metastasis models were used after priming the animals with exosomes to study the role of exo-AnxA2 in breast cancer metastasis. Results: Characterization of exo-AnxA2 by Western blot and AFM revealed that it is highly expressed in cancer exosomes than exosomes from normal cells. In vitro and in vivo angiogenesis studies revealed exo-AnxA2 to be a potent inducer of angiogenesis and inhibition of exo-AnxA2 significantly inhibits angiogenesis (~3 fold and ~5 fold decrease with LCKLSL vs. control in endothelial assay and matrigel plug assay respectively). Tail vein and intracardiac metastasis models showed that exo-AnxA2 promotes lung and brain metastasis. Knockdown of exo-AnxA2 showed ~2 fold decrease in lung and brain metastasis vs. control respectively, as evident from bioluminescence imaging. Conclusion: We found that exo-AnxA2 correlates positively with breast cancer progression. Furthermore, we found that exo-AnxA2 is a potent inducer of angiogenesis and breast cancer metastasis indicating a possible role of exo-AnxA2 in tumor- microenvironment signaling and cancer progression.