Movement of cancer cells from the primary tumor to distant metastatic sites represents the most lethal stage of cancer progression. The potential for a cell to migrate and invade depends on a multitude of signaling pathways, which present novel targets for anti-cancer therapies. Reactive Oxygen Species (ROS) are known modulators of tumorigenicity and act as novel second messengers in a multitude of cellular signaling cascades. Intrinsic levels of ROS are elevated in numerous metastatic cancer types. The ability of tumor cells to survive and thrive with an increased ROS milieu suggests that these have evolved to utilize changes in sublethal ROS levels to drive pro-metastatic signaling events. Our preliminary data suggest that picomolar sublethal increases in mitochondrial derived ROS/hydrogen peroxide (H2O2) lead to a more tumorigenic, migratory and invasive phenotype in metastatic cancer cells, in vitro and in vivo. These changes in ROS lead to alterations in pro-migratory signaling cascades, following oxidation and inactivation of key phosphatases. In addition, these studies present a novel concept that H2O2 is a regulatory factor that essentially has opposing effects on membrane localization of pro- and anti- migratory signaling players. We therefore hypothesize that sub lethal increases in mitochondrial ROS production regulate the spatial activation of pro-migratory signaling pathways enhancing the metastatic potential of cancer cells. Using a metastatic bladder cancer model this will be tested by: 1) Investigating the cellular site of action of mitochondrial ROS and their role in regulating oxidation and spatial distribution of Protein Tyrosine Phosphatases (PTPs) involved in migratory signaling. 2) Determining the effects of mitochondrial ROS on mediators of Focal Adhesion Kinase signaling during migration;and 3) Assessing the consequences of enhanced intracellular ROS levels in an in vivo model of metastatic bladder cancer and the effectiveness of antioxidant adjuvant therapy in this model. Importantly, these studies will establish a novel role for ROS as second messengers in regulating the spatial activation of pro-migratory signaling pathways, further our understanding of the role of mitochondrial ROS in metastatic progression;and determine the therapeutic potential of antioxidants, targeted to inhibit pro-migratory signaling, in metastatic bladder cancer. PUBLIC HEALTH RELEVANCE: This research will further our knowledge of the intracellular mechanisms that regulate migration and invasion of tumor cells. Understanding the role of reactive oxygen species in these events will aid in the development of novel antioxidant based cancer therapies targeting metastasis.