Tumor-derived exosomes are emerging mediators of tumorigenesis and tissue-specific metastasis. Proteomic profiling has identified Annexin II as one of the most highly expressed proteins in exosomes; however, studies focused on the biological role of exosomal Annexin II (exo-Anx II) are still lacking. In this study, mechanistic insight was sought regarding exo-Anx II and its function in angiogenesis and breast cancer metastasis. Multiple in vitro and in vivo techniques were used to study the role of exo-Anx II in angiogenesis. Using atomic force microscopy and Western blotting, exo-Anx II expression was characterized in normal and breast cancer cells. In addition, organ-specific metastatic breast cancer cells and animal models were used to define the role exo-Anx II in breast cancer metastasis. Results revealed that exo-Anx II expression is significantly higher in malignant cells than normal and premetastatic breast cancer cells. In vitro and in vivo studies demonstrated that exo-Anx II promotes tPA-dependent angiogenesis. Furthermore, in vivo analysis indicated that metastatic exosomes create a favorable microenvironment for metastasis, and exo-Anx II plays an important role in this process, as priming with Anx II-depleted exosomes reduces brain (∼4-fold) and lung (∼2-fold) metastasis. Upon delineating the mechanism, it was discovered that exo-Anx II causes macrophage-mediated activation of the p38MAPK, NF-κB, and STAT3 pathways and increased secretion of IL6 and TNFα. These data demonstrate an important role for exo-Anx II in breast cancer pathogenesis.Implications: Exosome-associated Annexin II plays an important role in angiogenesis and breast cancer metastasis, which can be exploited as a potential biomarker as well as a therapeutic target for diagnosis and treatment of metastatic breast cancer. Mol Cancer Res; 15(1); 93–105. ©2016 AACR.

Increasing evidence suggests that AnxA2 contributes to invasion and metastasis of breast cancer. However, the clinical significance of AnxA2 expression in breast cancer has not been reported. The expression of AnxA2 in cell lines, tumor tissues, and serum samples of breast cancer patients were analyzed by immunoblotting, immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. We found that AnxA2 was significantly upregulated in tumor tissues and serum samples of breast cancer patients compared with normal controls. The high expression of serum AnxA2 was significantly associated with tumor grades and poor survival of the breast cancer patients. Based on molecular subtypes, AnxA2 expression was significantly elevated in tumor tissues and serum samples of triple-negative breast cancer (TNBC) patients compared with other breast cancer subtypes. Our analyses on breast cancer cell lines demonstrated that secretion of AnxA2 is associated with its tyrosine 23 (Tyr23) phosphorylation in cells. The expression of non-phosphomimetic mutant of AnxA2 in HCC1395 cells inhibits its secretion from cells compared to wild-type AnxA2, which further suggest that Tyr23 phosphorylation is a critical step for AnxA2 secretion from TNBC cells. Our analysis of AnxA2 phosphorylation in clinical samples further confirmed that the phosphorylation of AnxA2 at Tyr23 was high in tumor tissues of TNBC patients compared to matched adjacent non-tumorigenic breast tissues. Furthermore, we observed that the diagnostic value of serum AnxA2 was significantly high in TNBC compared with other breast cancer subtypes. These findings suggest that serum AnxA2 concentration could be a potential diagnostic biomarker for TNBC patients.