Project Summary/Abstract There is a gap in our understanding of how hormone prolactin (PRL), secreted by adipocytes, and the serine/threonine PAK1 connect to coordinately regulate progression of human breast cancer (BC) and adipocyte-breast cancer cell crosstalk. The long-term goal of this research is to establish PAK1, activated by adipocyte-secreted PRL, as a new diagnostic tool for the metastatic potential of BC cells and provide needed insight into the underlying mechanisms. The overall objective of the current application is to establish an experimental system to study effects of adipocyte-secreted PRL on breast cancer cells and adipocyte-breast cancer cells crosstalk and further establish the mechanisms by which PRL-activated PAK1 contributes to breast cancer. The central hypothesis is that that adipocyte-produced prolactin enhances invasiveness, tumorigenicity and metastatic potential of breast cancer cells through activation of PAK1 kinase and pTyr- PAK1 is a biomarker to predict metastatic potential of these breast cancer cells. The rational for the proposed research is that breast adipocytes secret adipocytokines including PRL, which may influence breast tumor behavior. PRL promotes JAK2 activation and tyrosyl phosphorylation of PAK1 in breast cancer cells. The central hypothesis will be tested by pursuing the following specific aims: Specific Aim 1 will test our working hypothesis that PAK1 activated by adipocyte-derived PRL enhances breast cancer cell invasiveness and cell motility via Tec kinase and that adipocyte-breast cancer cells crosstalk induces secretion of MMP-11 by adipocytes and MMP-1 by breast cancer cells. Specific Aim 2 will test our working hypothesis that PAK1 activated by adipocyte-derived PRL affects tumorigenicity and metastatic potential of breast cancer cells in vivo and that pTyr-PAK1 can serve as a biomarker to predict breast tumor metastasis. Under the sub-aim, characterized PAK1 phospho-specific antibodies will be used to evaluate metastatic potential of the cells at an early stage of BC. This proposal is conceptually innovative because it is expected to vertically advance and expand our understanding of how obesity through adipocyte-secreted PRL influences BC and the unique role of PRL-activated PAK1 in this process. This proposal is technically innovative as well because it will employ unique PAK1 phospho-specific antibodies. The proposed research is significant because the elucidation of PRL/PAK1 mediated intracellular signaling pathways will enhance our understanding of the mechanisms involved in regulation of BC and will lead to development of PAK1-based tools for BC diagnosis.