The spread of breast cancer to other organs such as the liver, lungs and bone, is frequent in patients with advanced forms of the disease, and still accounts for the majority of deaths from breast cancer. Several factors that modulate actin cytoskeleton dynamics are key determinants of metastatic disease among which are members of the annexin family of Ca2+-dependent membrane binding proteins. One of these proteins, annexin A6 (AnxA6) has been shown to interact with membranes with slightly different kinetics. Its association with the cell membrane inhibits Ca2+ influx and cell proliferation; and a recent report revealed that AnxA6-depleted MDA-MB-436 invasive breast cancer cells grew in anchorage-independent manner. This SC2 proposal is based on these observations and our preliminary data suggesting that AnxA6 expression positively correlates with the motile/invasive phenotype of breast cancer and that loss of AnxA6 expression may be a critical step in the switch from anchorage-dependent to anchorage-independent cell growth that is typical of tumor growth. However, the mechanisms by which AnxA6 influences breast cancer progression and whether our in vitro observations can to be reproduced in vivo remain unknown. The overall hypothesis is that extracellular Ca2+- induced cell membrane-associated AnxA6 promotes the spread of invasive/motile breast cancer cells to distant organs by facilitating their interaction wth other cells and with the surrounding extracellular matrix. This will be tested using two specific aims: 1) to assess whether AnxA6 is essential for the metastasis of motile/invasive breast cancer cells to distant organs in vivo, and 2) to determine whether the establishment and growth of invasive breast cancer cells in high Ca2+ microenvironments require AnxA6. The proposed studies will not only determine the role of AnxA6 in breast cancer metastasis and tumor progression but will also examine how AnxA6-dependent Ca2+ handling mechanisms could be exploited to assess AnxA6 as a therapeutic target for the prevention and/or treatment of metastatic breast cancer.