Molecular mechanisms controlling metastasis in breast cancer are not well understood. During invasion of the extracellular matrix (ECM) cancer cells alternate between fast, amoeboid, and slow mesenchymal types of movement. This plasticity in movement is essential for efficient invasion, but it is not well understood how it is controlled. We have found that the RhoGEF Net1A is an important regulator of invasion plasticity in breast cancer cells, stimulating amoeboid and suppressing mesenchymal movement. Net1A regulates this process by controlling RhoA and deacetylase function to impact cytoskeletal organization and gene expression. Net1A is exceptional among RhoGEFs in that it is sequestered in the nucleus to prevent aberrant RhoA activation, and it is clear that subcellular localization is a key determinant of the oncogenic potential of Net1 isoforms. Recently we have shown that Rac1 activation downstream of integrin or growth factor receptor activation stimulates Net1A relocalization to the plasma membrane. We have also found that Net1A relocalization is maintained by site-specific acetylation, and that the actions of specific deacetylases are critical to suppressing Net1A activity towards RhoA. Moreover, we have observed that Net1A reciprocally controls the activity of specific deacetylases to regulate cytoskeletal organization and expression of key genes associated with cell invasion, such as MT1-MMP. These findings support our hypothesis that that Net1A functions as a nodal point to temporally and spatially regulate RhoA activation and deacetylase activity which underlie breast cancer cell motility, invasion and metastasis. In this proposal we will use a combination of cell-based and in vivo assays to understand how Net1A localization is regulated to drive RhoA activation and reciprocally control deacetylase activity during ECM invasion. We will also determine how Net1 isoforms contribute to mammary gland tumorigenesis and metastasis in vivo. In Aim 1 we will demonstrate how EGFR and HER2 activation stimulates Net1A export from the nucleus to stimulate cell motility and ECM invasion. We will demonstrate how Net1A acetylation affects its function and identify the deacetylases that regulate Net1A. In Aim 2 we will determine how Net1A controls HDAC and Sirtuin function to regulate acetylation of cytoskeletal organizing proteins and gene expression. In Aim 3 we will identify the molecular basis for how genetic deletion of Net1 affects mammary gland development in mice, and determine whether Net1 is required for HER2/Neu driven mammary gland tumorigenesis and metastasis. Completion of these aims will provide a mechanistic understanding of how Net1 isoforms contribute to breast cancer cell motility and invasive potential, and will provide novel targets for intervention in metastatic disease.