S100A14Protein Binds HER2and Modulates HER2Signaling

S100protein is a calcium-binding protein family, which has been implicated in a variety of important physiological functions. Deregulated expression of these proteins has been reported in different types of neoplasia and many S100proteins are correlated with tumor development or progression. S100A14is a new member of the S100family which was identified in2002, its functional role in tumor development is poorly understood. Our previous study showed that the expression level S100A14was significantly down-regulated in esophageal squamous cell carcinoma (ESCC) and there was a positive correlation between the expression levels of S100A14with lymph node metastasis. S100A14promotes cell motility and invasiveness by regulating the expression and function of MMP2in a p53-dependent manner and extracellular S100A14binds to RAGE and promots cell proliferation through stimulating MAPK and NF-κB signaling cascades. In order to further characterize expression pattern of S100A14and the relationship with clinicopathological features, immunohistochemistry analysis were performed in primary tumors and matched normal tissues. The results indicated that the expression of S100A14was obviously upregulated in breast cancer samples compared with matched normal counterparts. Statistical analysis revealed that there was a significant correlation between the expression levels of S100A14and HER2(r=0.425, P <0.001). Moreover, S100A14and HER2were colocalized in plasma membrane of most breast cancer tissues, then immunofluorescence microscopy were performed to show the cellular localization of S100A14and HER2in breast cancer cell lines BT474and SK-BR-3. The results showed that both the S100A14and HER2were colocalized in plasma membrane. Based on the above results, we speculate that S100A14may interact with HER2, and then co-immunoprecipitation and pull down analysis were performed. The results clearly showed that S100A14interacts with the intracellular domain of HER2(HER2-ICD) but not the extracellular domain. To investigate the sites in S100A14and HER2necessary for HER2-S100A14binding, we constructed four HER2-ICD truncation mutants and four S100A14truncation mutants. The results indicated that residues956-1154of HER2intracellular domain and the C-terminal EF hand of S100A14are essential for the two proteins binding.To further investigate the function of S100A14in human breast cancer cells, siRNA-mediated silencing in MCF-7cells was performed. The plasmid expressing Flag-HER2was transfected into S100A14-kncockdown MCF-7cells, and then Western blot was performed to evaluate HER2downstream signal molecules. We observed a decrease of HER2phosphorylation, signaling, and also HER2-stimulated cell proliferation in S100A14-knockdown MCF-7cells by MTS assay.Several members of the S100family can be secreted into serum. A variety of S100members, including S100A4, S100A8, S100A9, were taken as the serum tumor markers in diagnosis and prognosis of the tumor. Our previous studies indicated that S100A14can be secreted into the supernatant of cultured tumor cells and extracellular S100A14proteins have important functions. Comparing S100A14serum levels of125cases of breast cancer with75healthy controls and44patients suffering from benign breast lesions, we found significantly increased S100A14serum levels in breast cancer patients in comparison with healthy controls or patients suffering from benign breast lesions (benign tumor or adenosis of breast)(P<0.001).Taken together, we conclude that S100A14may play an important role in breast cancer progression through regulating HER2signaling by interacting with HER2. Our findings add a new dimension to S100A14function as a modulator of cell signaling and provide mechanistic evidence for its role in breast cancer progression.