PROJECT SUMMARY Radiation therapy (RT) is widely used to treat localized prostate cancer (PCa). However, cancer cells often develop resistance to RT through unknown mechanisms, resulting in cancer recurrence. To improve RT, there is a dire need to uncover cellular events that cause cells to become resistant. We previously demonstrated that PCa heterogeneity, particularly in prostate cancers with an abundant mitochondria subpopulation, often survive and regrow after RT (termed radiation resistant prostate cancer, or RR-PCa). Elevation of mitochondrial mass, number, reactive oxygen species (ROS), and biogenesis markers is acquired in RR-PCa cells. We further demonstrated that knockdown of the mitochondrial biogenesis regulator, TFAM (transcription factor A, mitochondrial), significantly restored the sensitivity of RR-PCa cells to RT. Hence, our overarching hypothesis is that RT-activated mitochondrial biogenesis, via ROS, is an acquisition mechanism that drives PCa survival post- RT, a premise that will undergo stringent examination in the proposed studies. ROS are known to directly and indirectly regulate mitochondrial homeostasis through fusion, fission, mitophagy, and biogenesis. We screened FDA-approved drugs in search of compounds that are nontoxic to normal cells and have the ability to raise the level of mitochondrial hydrogen peroxide (mtH2O2) in PCa cells while blocking mitochondrial protein translation. We found azithromycin (AZM), a macrolide antibiotic, to be an effective prototype compound that possesses both properties. We further demonstrated that AZM combined with RT enhances the death of PCa cells with an abundant mitochondrial subpopulation, compared to AZM or RT alone. Thus, we propose to advance our findings and identify the mechanism(s) that effectively inhibit the survival of post-irradiated cancer cells, to improve RT efficacy. The specific aims are: 1) to define the molecular mechanism(s) by which RT-activated mitochondrial biogenesis promotes cell survival and metabolic adaptations of PCa cells with abundant mitochondria, both in vitro and in vivo; 2) to determine if overloading mtH2O2 to target inherent mitochondria and RT-acquired mitochondria while blocking mitochondrial protein translation in RT-acquired mitochondria enhances radiosensitivity of RR-PCa cells, and 3) to improve RT using a mtH2O2 generator and a mitochondrial protein translation inhibitor, AZM as prototype, in an orthotopic mouse xenograft model and a patient-derived xenograft model of PCa with activated mitochondrial biogenesis. This study uses state-of-the-art platforms including the reverse phase protein array, stable isotope-resolved metabolomics, super-resolution microscopy with Imaris software, TEMPOL-enhanced MRI imaging, and a high resolution O2k-FluoRespirometer. The proposed studies are expected to uncover novel molecular insights by which concurrently targeting mitochondrial redox capacity and mitochondrial biogenesis improve RT efficacy of RR-PCa.

PROJECT NARRATIVE Radiation-resistant prostate cancer (RR-PCa) is highly unpredictable which frequently results in cancer recurrence in many patients. This project aims to 1) establish the underlying mechanism(s) of mitochondrial biogenesis that contribute to RR-PCa cell survival and metabolism and 2) determine if targeting inherent mitochondria and radiation-acquired mitochondria, can overcome RR-PCa. The successful completion of this project will establish a new and significant therapeutic platform for making radiation therapy more effective.