This proposal builds on prior studies to define the mechanisms responsible for the direct antitumor effects of the attenuated mycobacterium bovis, Bacillus Calmette-Gu?rin (BCG). Intravesical BCG remains the standard of care treatment for patients with high risk, nonmuscle-invasive bladder cancer (urothelial carcinoma, UC). Unfortunately, BCG has significant limitations. Only 65% of the patients receiving treatment respond to this agent. Toxicity resulting from BCG's infectious potential limits its use to high-risk patients. With the goal of improving treatment efficacy, and/or decreasing treatment toxicity, the investigators have defined a robust set of intermediate endpoints for measuring BCG's direct effect on human UC cells. These endpoints include activation of intracellular signaling pathways, and transactivation of the genes for multiple chemokines, cytokines and white cell receptors. Signal activation/gene expression culminates in phenotypic changes characterized by cell-cycle arrest and apoptotic resistance. Subpopulations of cells undergo necrosis releasing the necrosis related chemokine HMGB1. As shown in the previous funding interval HMGB1 release is required for the in vivo antitumor response to BCG. The group's panel of endpoints provides insight into BCG's mechanism of action, and serves as a toolbox for assessing strategies to improve treatment outcomes. The nature of the direct cellular response to BCG has led the researchers to hypothesize that cellular oxidative stress (COS), elicited by BCG binding/internalization, is a critical component of BCG's antitumor efficacy. Our published and preliminary data demonstrates that BCG serves as a free radical generator. Loss of BCG viability diminishes free radical production and the UC cell response to BCG across all intermediate endpoints. Downstream of the cellular binding/internalization of viable BCG, the researchers have demonstrated that transactivation of specific oxidative stress pathways and associated free radical production also are required for the optimal UC cell response to BCG. Pharmacologic enhancement of these pathways/free radicals potentiates the response of UC cells to viable and nonviable BCG. This proposal will test the hypothesis that viable BCG's pharmacogenetic effects on UC cells, a requirement for antitumor activity, are a direct consequence of BCG generated H2O2 which serves as the trigger for COS and cell damage. Production of H2O2 by BCG, following binding to and internalization by UC cells, sets the stage for a second wave of cell-generated oxidants involving iNOS. Free radicals (superoxide and NO), and reactive molecules potentiate intracellular signaling pathways and downstream gene expression and result in a cellular phenotype that defines the BCG-treatment effect. Loss of BCG viability is associated with decreased H2O2 production, inefficient induction of COS and its direct consequences, and decreased treatment efficacy. Manipulation of contributors to the COS response to BCG represents an opportunity to enhance COS activity for greater antitumor effect. In combination with viable BCG such approaches afford an opportunity for greater treatment efficacy. When used with heat killed BCG these approaches promise to maintain treatment efficacy while decreasing the potential toxicity associated with viable BCG. In addressing this hypothesis the proposed studies will meet two fundamental goals. First, the researchers will build upon existing data to complete their understanding of the factors contributing to BCG induced COS and COS associated cellular injury. By understanding the specific COS pathways activated in response to BCG, and the downstream effects of those pathways, the group will be poised to address their second goal. Specifically, to employ robust in vitro and in vivo systems to test strategies to potentiate BCG induced oxidant generation and oxidative damage in UC cells for improved treatment efficacy and/or decreased treatment toxicity. Selected treatment strategies will be translatable to the clinic in a way that will result in decreased tumor recurrence and progression rates and broader therapeutic applicability of BCG.