Phosphorylation of Annexin A2 is Essential for its Association with Exosomes in Triple Negative Breast Cancer

Abstract

Introduction: Studying triple negative breast cancer (TNBC) is the utmost importance 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. Also, N-terminal phosphorylation of AnxA2 at Tyrosine (Tyr) 23 has been implicated in several cancer progression. However, the mechanism through which AnxA2 enters into the exosomes has not been elucidated in TNBC. Methods: Plasmids expressing constitutive phosphomimetic AnxA2 (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, invasion and proliferation. 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 was analyzed on invasiveness and proliferation in cancer cells. Results: Our results showed that MDA-MB-231 TNBC cells expressing phosphomimetic AnxA2 showed increased migratory, invasive and proliferative capacity compared to cells expressing non-phosphomimetic AnxA2. Exosomes derived from phosphomimetic cells had increased AnxA2 expression at surface compared to exosomes derived from non-phosphomimetic cells. In addition, high surface expression of AnxA2 in exosomes derived from phosphomimetic cells induced invasive and proliferative characteristics in CAL-148 breast adenocarcinoma cells compared to exosomes derived from non-phosphomimetic cells (exo-AnxA2-Y23F). Conclusion: Phosphorylation of AnxA2 at Tyr23 plays an important role in imparting metastatic phenotype to the TNBC cells. In addition, the phosphorylation of AnxA2 at Tyr23 is an important event for its entry into the exosomes that promotes invasion and proliferation in cancer cells.