Project Summary Two thirds of breast tumors express estrogen receptor (ERα), and although many initially respond to endocrine therapy, a large fraction subsequently develop resistance, causing death due to advanced hormone-resistant disease. A number of groups, including ours, have recently documented the occurrence of single nucleotide mutations in the ERα gene (ESR1) in 20-30% of endocrine-resistant metastatic breast cancer. Limited clinical evidence suggests that patients whose tumors have gained ESR1 mutations suffer from shorter survival. ESR1 hotspot mutations cluster in the ligand-binding domain. Analysis of our CRISPR/Cas9 genome edited breast cancer cells with the most common ESR1 mutations (Y537S and D538G) showed ligand-independent transcriptional activity, and partial resistance to selective estrogen receptor modulators (SERMs) and downregulators (SERDs). Mutant ERα also shows regulation of genes not classically regulated by estrogen, with significant differences between D538G and Y537S. Genes uniquely regulated by mutant ERα are involved in motility, migration, and adhesion, and we have identified such gain-of-function phenotypes in the ESR1- mutant cells. We hypothesize that mutations in ESR1 are enriched in endocrine-resistant breast cancer due to ligand-independent activity of mutant ERα, and a unique gain of function regulating motility and adhesion. The altered transcriptional activities of mutant ERα are mediated by reprograming of the ERα cistrome, as a result of altered interaction with coregulators. Finally, we hypothesize that SERDs will be most effective in inhibiting mutant ERα driven tumor growth. To address these hypotheses we will use a multidisciplinary approach including unique in vitro and in vivo models of mutant ERα action, and analysis of clinical specimens. The specific aims are 1) Evaluate how different ESR1 mutations alter its transcriptional activity and function, 2) Characterize the ligand-dependent and drug-resistant activities of mutant ERa, focusing on gain of function activities, and 3) Determine whether the unique transcriptional regulation by mutant ERα is present in advanced endocrine resistant breast cancers, and whether it is critical for progression and metastasis. We expect that our comprehensive structure-function studies of ERα mutations will not only provide basic information regarding hormone resistance, but will highlight novel routes to therapeutic targeting. !