Diversity Supplement for Kahleel Guerrier: Targeting Mitochondrial Redox Capacity to Overcome Cancer Subtype that Regrowth After Radiation

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. In order to improve RT, there is a dire need to uncover the cellular events that cause cells to become resistant. Through our in-depth investigations we demonstrated that PCa heterogeneity, particularly prostate cancers with an abundant mitochondria subpopulation, often survive and regrow after RT (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 compared to radiosensitive PCa. 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 activation of mitochondrial biogenesis, via ROS, is an acquisition mechanism that drives PCa survival of 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 the most 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. Encouraged by these robust results, we aim to advance our findings and identify the mechanism(s) that effectively inhibit the survival of post-irradiated cancer cells, to improve RT efficacy. The goals are: 1) to determine 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 investigate if overloading mtH2O2 to target inherent mitochondria and RT-acquired mitochondria while blocking mitochondrial protein translation in RT-acquired mitochondria enhances radiosensitivity of PCa cells with abundant mitochondria, 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 abundant mitochondria. RR-PCa tumor overexpressing inducible mitochondrial catalase, which rapidly removes H2O2, will be used to validate improved RT efficacy by AZM-generated mtH2O2. 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.