Overexpression of the receptor tyrosine kinase HER2 (ErbB2) and activation of transforming growth factor ??(TGF?) signaling are both associated with progression of breast cancers to a more metastatic phenotype. Overexpression of the HER2 proto-oncogene is permissive for the tumor promoting effects of TGF?. The objective of this proposal is to study the reciprocal, temporal and spatial crosstalk between TGF? and HER2, which involves not only intra-/intercellular but also microenvironmental events. We hypothesize that in HER2-mediated transformation, TGF? can 1) dynamically modulate HER2 subcellular distribution and the repertoire of HER2 cellular substrates/signal transducers;2) regulate the magnitude and amplitude of HER2 signaling through the modulation of HER2 phosphorylation and cellular trafficking;3) modify the ECM and stroma to create a microenvironment that facilitates tumor growth and metastasis;and 4) attenuate the response of HER2-dependent cells to therapeutic inhibitors of HER2. Understanding the temporal and spatial features of the crosstalk between TGF? and HER2 signaling will disclose new therapeutic targets, as well as potentially allow the optimization of current therapeutic inhibitors of HER2. The results will also shed light on the perspective of a combined therapy against TGF? and HER2 in breast cancer patients. The following specific aims will be pursued. Specific aim 1: to determine whether activated TGF? receptors spatially and temporally modulate HER2 signaling in mammary epithelial cells and how TGF? -induced cytoskeleton remodeling affects HER2 distribution, trafficking and signaling. Specific aim 2: to determine whether the Rac/Pak pathway is required for the invasiveness and survival mediated by the interaction between HER2 and TGF?, whether HER2 is a substrate of Pak1 and whether HER2 colocalizes with Pak1 and active Rac1 in primary breast tumors. Specific aim 3: to determine whether TGF? and HER2 modify microenvironmental components by utilizing proteomic approaches and whether these contribute to tumorigenesis in mice. Specific aim 4: to determine whether activated TGF? signaling can confer resistance to therapeutic inhibitors of HER2 in mammary cancer cells and the role of PI3K and Rac/Pak pathways in TGF? -conferred resistance.