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.