Early prediction of metastatic breast cancer is important for improvement of prognosis and survival rate. The present study aimed to identify secreted protein biomarkers for detection of invasive breast cancer. To this end, we performed a comparative proteomic analysis by a combination of 2DE and MALDI-TOF MS analysis of conditioned media from invasive H-Ras MCF10A human breast epithelial cells and noninvasive MCF10A and N-Ras MCF10A cells. We identified a list of 25 proteins that were strongly detected in media of H-Ras MCF10A and focused on annexin II, which was shown to be involved in cell motility. Invasive triple-negative human breast carcinoma cells, Hs578T, and MDA-MB-231, showed increased levels of annexin II in media, demonstrating that secretion of annexin II correlated well with the invasive phenotype of cells. We demonstrated a crucial role of annexin II in breast cell invasion/migration and actin cytoskeleton reorganization required for filopodia formation. Annexin II levels in the plasma samples and breast cancer tissues of breast cancer patients were significantly higher than those of normal groups, providing a clinical relevance to our in vitro findings. Taken together, we identified annexin II as a novel secretory biomarker candidate for invasive breast cancer, especially estrogen receptor-negative breast cancer.
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