Project Summary/Abstract. Non-alcoholic fatty liver disease can make the liver permissive to liver metastasis. Patients taking an androgen signaling inhibitor, such as enzalutamide for the treatment of prostate cancer can experience similar biologic changes to the liver. These systemic changes in the patient lipid profile are associated with liver injury can result in a quantifiable acceleration of biologic aging of the liver tissue. Prostate cancer cells acquire genetic adaptations that enable survival in the liver as a metastatic niche. We found that enzalutamide exposure promoted expression of BMP ligands in prostate cancer epithelial cells in a glutamine signaling-dependent manner. Enzalutamide also increased expression of SLC1A5, a glutamine transporter able to regulate tumor growth. Prostate cancer epithelial cells grown without glutamine had significantly lower levels of BMP ligands compared to cells treated with glutamine, glutamate or enzalutamide. Glutamine inhibition was found to block expression of SLC1A5 in prostate epithelia co-cultured with prostate cancer-associated fibroblasts. The mechanism of transporting glutamine into the epithelial cells was sufficiently blocked using these inhibitors, and fibroblast-specific genes were also affected. Wnt3a expression and glutamine secretion in prostate cancer- associated fibroblasts increased after enzalutamide but decreased with glutamine inhibition. Based on these novel findings, we tested if glutamine inhibition could be effective on the prostate tumor microenvironment, where prostate cancer epithelia and cancer-associated fibroblasts were co-cultured. We found that enzalutamide increased neuroendocrine differentiation in prostate epithelia, abrogated when co-cultures were treated with glutamine inhibitor alone or in combination with enzalutamide. The potential role of enzalutamide on methylation markers associated with biologic aging was observed where prostate epithelia treated with enzalutamide had increased promoter enrichment of TET2 on its downstream target TBX2; glutamine inhibition decreased promoter enrichment. We hypothesize that biologic aging of the liver induced by high fat diet impacts the hepatic microenvironment to permit prostate metastasis. In Aim 1, we will define epigenetic changes in the liver that occur as a result of androgen signaling inhibition using enzalutamide. We will determine how high fat diet influences methylation patterns in the liver in a tumor-free environment. In Aim 2, we will identify the role of biologic aging in the liver induced by high fat diet or enzalutamide treatment after primary tumor expansion occurs. We found that prostate cancer epithelial expression of TET2, a methylcytosine dioxygenase mutated in cancers, is increased with enzalutamide or glutamine treatments. We will explore the androgen-TET2 crosstalk in prostate cancer epithelia as an adaptation promoting liver metastasis. The mechanism by which high fat diet and liver aging contrib...