Phosphorylation of Annexin A2 at Tyrosine 23 is Essential for its Association with Exosomes and for Imparting Invasive Phenotype to Breast Cancer Cells

Introduction: Studying triple negative breast cancer (TNBC) is important as treatment lacks targeted-based therapies. High expression of exosomal Annexin A2 (AnxA2), a Ca+2-dependent phospholipid binding protein, plays an important role in pre-metastatic niche formation and promoting cancer metastasis in TNBC. N-terminal phosphorylation of AnxA2 at Tyrosine(Y)23 has been implicated in several cancer progression. However, the mechanism through which AnxA2 associates with the exosomes and helps impart invasiveness to TNBC cells has been least elucidated. Methods: Plasmids expressing constitutive phosphomimetic (AnxA2-Y23E) and non-phosphomimetic AnxA2 (AnxA2-Y23F) mutant gene were transfected in MDA-MB-231 cells. Transfected cells were functionally validated for AnxA2 specific functions like migration and invasion. Exosomes isolated from AnxA2-Y23E (exo-AnxA2-Y23E) and AnxA2-Y23F (exo-AnxA2-Y23F) mutant cells were analyzed for surface expression of AnxA2. Effect of exosomes containing AnxA2-Y23E and AnxA2-Y23F mutant proteins were analyzed on invasiveness of cancer cells. Results: Our results showed that MDA-MB-231 TNBC cells expressing AnxA2-Y23E showed increased migratory and invasive capacity compared to cells expressing AnxA2-Y23F. Exosomes derived from AnxA2-Y23E cells had increased surface AnxA2 expression compared to exosomes derived from AnxA2-Y23F cells. High surface expression of AnxA2 in exosomes derived from AnxA2-Y23E cells induced invasive characteristics in CAL-148 breast adenocarcinoma cells compared to exosomes derived from AnxA2-Y23F cells. Conclusion: Phosphorylation of AnxA2 at Y23 plays an important role in associating AnxA2 with the exosomes which promotes invasiveness in cancer cells.