The purpose of this study was to address the role of ESR1 hormone-binding 
mutations in breast cancer. Soft agar anchorage-independent growth assay, 
Western blot, ERE reporter transactivation assay, proximity ligation assay 
(PLA), coimmunoprecipitation assay, silencing assay, digital droplet PCR 
(ddPCR), Kaplan-Meier analysis, and statistical analysis. It is now generally 
accepted that estrogen receptor (ESR1) mutations occur frequently in metastatic 
breast cancers; however, we do not yet know how to best treat these patients. We 
have modeled the three most frequent hormone-binding ESR1 (HBD-ESR1) mutations 
(Y537N, Y537S, and D538G) using stable lentiviral transduction in human breast 
cancer cell lines. Effects on growth were examined in response to hormonal and 
targeted agents, and mutation-specific changes were studied using microarray and 
Western blot analysis. We determined that the HBD-ESR1 mutations alter 
anti-proliferative effects to tamoxifen (Tam), due to cell-intrinsic changes in 
activation of the insulin-like growth factor receptor (IGF1R) signaling pathway 
and levels of PIK3R1/PIK3R3. The selective estrogen receptor degrader, 
fulvestrant, significantly reduced the anchorage-independent growth of ESR1 
mutant-expressing cells, while combination treatments with the mTOR inhibitor 
everolimus, or an inhibitor blocking IGF1R, and the insulin receptor 
significantly enhanced anti-proliferative responses. Using digital drop (dd) 
PCR, we identified mutations at high frequencies ranging from 12 % for Y537N, 
5 % for Y537S, and 2 % for D538G in archived primary breast tumors from women 
treated with adjuvant mono-tamoxifen therapy. The HBD-ESR1 mutations were not 
associated with recurrence-free or overall survival in response in this patient 
cohort and suggest that knowledge of other cell-intrinsic factors in combination 
with ESR1 mutation status will be needed determine anti-proliferative responses 
to Tam.