PROJECT SUMMARY Autophagy is a tightly regulated intracellular degradation and recycling process crucial for cellular homeostasis and adaptation to diverse cancer-relevant stresses. Because autophagy promotes the survival and metabolic fitness of established tumors, there is great interest in targeting autophagy to treat cancer. Importantly, anti- malarials, such as hydroxychloroquine (HCQ) are currently being repurposed as autophagy inhibitors in clinical oncology trials. However, inhibiting autophagy also results in aberrant accumulation of autophagy cargo receptors (ACRs), adaptor proteins that mediate the selective autophagic degradation of targets as well as function as multidomain signaling hubs. The accumulation of ACRs supports oncogenic progression, drives primary tumor growth and promotes therapeutic resistance in autophagy-deficient cells, but the role of ACRs during metastasis has been less clear. In recently published data, we have demonstrated that the accumulation of a specific ACR, called neighbor of BRCA1 (NBR1), promotes metastasis when autophagy is inhibited in tumor cells. In mouse mammary cancer models, genetic autophagy inhibition promotes spontaneous metastasis by enabling the outgrowth of disseminated tumor cells into overt macro-metastases. Furthermore, at both primary and metastatic sites, genetic autophagy inhibition leads to the marked expansion of tumor cells exhibiting aggressive and pro-metastatic basal epithelial differentiation, including the expression of cytokeratin 14 (CK14) and the transcription factor p63. The upregulation of NBR1 in autophagy-deficient tumors is both necessary and sufficient to promote metastatic outgrowth and pro-metastatic differentiation. Based on these findings, we hypothesize that autophagy inhibition promotes the emergence of multiple aggressive tumor subpopulations due to impaired NBR1 turnover. In aim 1, we will determine the mechanisms how NBR1 promotes basal epithelial differentiation in carcinoma cells. In aim 2, we will evaluate the effect of autophagy inhibition on tumor recurrence and intratumor heterogeneity. In aim 3, we will scrutinize how therapeutically modulating autophagy, both positively or negatively, impacts metastatic differentiation via promoting the turnover or sequestration of NBR1. Overall, this proposal will elucidate the mechanisms by which autophagy and NBR1 control aggressive differentiation programs in breast cancer and how this regulation impacts recurrence and treatment response.