Phosphorylation of Tyrosine 23 in Annexin A2 is Essential for its Association with Exosomes and for Imparting Invasive and Proliferative Capacity to other Cells




Chaudhary, Pankaj
Prakash Desai, Priyanka
Vishwanatha, Jamboor
Sun, Xiangle
Trivedi, Rucha


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Purpose: Triple negative breast cancer (TNBC) accounts for 15%-20% of all breast cancer cases. The lack of targeted-based therapies highlights the importance of studying TNBC. Elevated levels of Annexin A2 (AnxA2), a Ca+2-dependent phospholipid binding protein, has been correlated with worse overall survival in TNBC patients. Our previous data implicate that exosomal AnxA2 is involved in creating a pre-metastatic niche and facilitating metastasis in TNBC. Here, we demonstrated that phosphorylation of AnxA2 at Tyrosine23 (Tyr23) is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. Methods: pN1-EGFP plasmids expressing the phosphomimetic (AnxA2-Y23E) and non-phosphomimetic mutant (AnxA2-Y23F) gene at Tyr23 were overexpressed in MDA-MB-231 TNBC cells. Exosomes were isolated from the mutant cells by ultracentrifugation. Surface exosomal AnxA2 was detected by flowcytometry. CAL-148 cells were treated with exosomes to analyze invasion and proliferation by transwell invasion and proliferation assay, respectively. Transfer of exosomal AnxA2 (Exo-AnxA2) and signaling mechanism in CAL-148 were studied by immunofluorescence and Western blot, respectively. Results: Exo-AnxA2-Y23E-GFP had elevated surface AnxA2 expression compared to exo-AnxA2-Y23F-GFP. CAL-148 cells treated with Exo-AnxA2-Y23E-GFP showed high invasive and proliferative characteristics, with a higher expression of p-AnxA2, p-Src and p-Paxillin. Conclusion: Phosphorylation of Tyr23 in AnxA2 in TNBC cells is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells.