Project Summary/Abstract Our studies will investigate mechanisms associated with aging-related prostate cancer progression through our investigations of the prostate-specific homeobox gene NKX3.1. Work by us and others has shown that NKX3.1 is essential for normal prostate differentiation, whereas its loss promotes prostate cancer during aging. NKX3.1 serves as a gatekeeper to protect the prostate epithelium from cancer-promoting insults including oxidative stress and inflammation. Conversely, NKX3.1 loss abrogates such protection, thereby accelerating cancer progression. Our recent studies and new Preliminary data show that the traditional functions of NKX3.1 as a nuclear transcription factor are augmented by non-nuclear functions that are also required for suppression of prostate cancer. Thus, in normal contexts, NKX3.1 is localized to the nucleus where it regulates nuclear target genes. However, in conditions of oxidative stress, NKX3.1 also becomes localized to mitochondria, where it regulates mitochondrial genes essential for oxidative phosphorylation (OXPHOS). Our Preliminary data further show that the consequences of NKX3.1 loss are compounded as cells acquire mitochondrial mutations, leading to aging-associated acceleration of prostate cancer. In particular, our analyses of mouse models having loss of function of Nkx3.1 combined with a defective PolgA gene that renders mutated mitochondrial DNA (Nkx3.1; PolgA mutant mice) reveals aging-related acceleration of prostate cancer with evident mitochondrial dysfunction, reduced OXPHOS activity, and hallmarks of aging, including cellular senescence and telomere attrition. Thus, our studies will examine the hypothesis that suppression of prostate cancer by NKX3.1 requires its nuclear and non-nuclear functions, and that NKX3.1 loss synergizes with mitochondrial dysfunction to promote prostate cancer during aging. Aim 1 will investigate the nuclear and non-nuclear functions of NKX3.1 in prostate cancer by leveraging