Metastasis, cancer spreading, is often a final and fatal stage in the progression of solid malignancies. Early detection of localized breast cancer results in a higher relative five-year survival rate (98%) while the relative five-year survival rae drops significantly (26%) for those women who have distant metastases. Therefore, a better understanding of mechanistic insight regarding the development and progression of this disease is of extreme importance in clinical setting. Our study revealed that cytokine CCL18 released from tumor-associated macrophage promotes breast cancer metastasis and serves as a novel indicator for poor survival in the clinical oncology (Chen et al., 2011. Cancer Cell). In addition, our recent study identified a breast cancer metastasis signaling network involving CCL18 and its potential effectors ARF6-ACAP4-ezrin. However, little is known regarding the molecular mechanism(s) by which CCL18 operates in breast cancer metastasis. The long-term goal of our research is to delineate how ARF6-ACAP4-ezrin interaction orchestrates stimulus-coupled breast cancer metastasis. To address this question, three Specific Aims are proposed: first, we will delineate the ARF6 activity gradient underlying breast cancer cell dynamics using a novel biosensor combined with ACAP4 small molecule inhibitors; second, we evaluate how phospho-ezrin interacts with ACAP4 using epitope-tagging, chemical foot-printing, and cross-linking approaches. These studies will involve a detailed analysis of the structural determinants that mediate a direct ezrin-ACAP4 contact. Binding domain data will be used to design peptides that potently and specifically perturb ezrin-ACAP4 interactions in in vitro binding assays. The importance of such an interaction in breast cancer invasion will then be evaluated by functional assay and supra-resolution imaging analysis. Third, we plan to investigate the potential mechanisms underlying ACAP4-mediated breast cancer interacts in animals. These studies will be facilitated by in vivo bioluminescence images coupled with small molecule inhibitor treatment in live xenografted animals. Studying the molecular mechanisms underlying breast cancer metastasis is of great significance in understanding the solid tumor progression, and is also expected to be of great benefit in leading to pharmacological strategies for preventing tumor cell spreading.