Project Summary The proposed research tests the novel concept that mitophagy and mitochondrial biogenesis are coordinately regulated to promote both turnover of old mitochondria but also to facilitate rapid and wholesale re-programming of mitochondria in response to stress. Furthermore, we suggest that this regulated coupling of mitophagy and mitochondrial biogenesis is disrupted in cancers by oncogenic signaling resulting in increased mitochondrial mass and incomplete metabolic switching in response to stress. Finally, we identify novel chemical regulators of these processes that may ultimately be exploited for cancer therapy. These ideas are tested in the aims set out below. In Aim 1, we set out to measure the kinetics and interdependence of mitophagy and mitochondrial biogenesis in normal breast epithelia compared to a panel of breast cancer cells, to allow us to identify pathways and commonalities in how these two processes are controlled. We also use proteomic approaches to define changes in mitochondria in response to stress and as a function of intact mitophagy or mitochondrial biogenesis. Finally, we develop approaches to ?track? mitochondria to determine whether there are ?stem? mitochondria that are resistant to mitophagy, to which new mitochondrial protein mass is preferentially added. In Aim 2, we leverage data from normal cells examined in Aim 1, and determine how these pathways and signaling events are altered in different human breast cancer cell lines and in primary human breast cancers. We examine whether increased mitochondrial mass is linked to defective mitophagy, increased biogenesis or potentially uncoupling of these process and whether this in turn is associated with specific oncogenic lesions. We use inducible c-Myc systems to address how oncogenic activity driving biogenesis affects coordination with mitophagy and overall mitochondrial mass and whether this plays into a tumor suppressor role for mitophagy and a tumor promoting role for biogenesis. In Aim 3, we identify of FDA approved drugs that promote tumor cell killing by preventing removal of damaged mitochondria, or the generation of new healthy mitochondria, with limited effects on normal cells. Since these drugs are already FDA approved for other purposes, they could be translated to the breast cancer clinic rapidly (within 2-5 years) tailored to their new role as inhibitors of mitochondria housekeeping, rapid tumor cell killing and promoting recurrence-free survival. Overall, we expect to provide insight to novel mechanisms of tumorigenesis that relate to control of mitophagy and mitochondrial biogenesis in such a way that may explain the heterogeneity in mitochondrial mass detected in primary human breast cancers.!