Impact of gain-of-function estrogen receptor alpha gene mutations on ¹⁸F-fluoroestradiol binding affinity.

Objectives: Mutations in the estrogen receptor alpha gene (ESR1) identified in patients with metastatic estrogen receptor alpha (ERα) positive breast cancer are associated with endocrine therapy resistance and reduced survival. The aim of this research was to assess if mutations in the ligand binding domain of ERα alter ¹⁸F-fluoroestradiol (FES) binding in breast cancer cells.

Methods: Stable cell lines were generated using ER negative MDA-MB-231 breast cancer cells to express either wild-type (WT) ER or various reported mutations within the receptor ligand binding domain (D538G, Y537C/N/S, E380Q, L536Q). ERα functional activity was determined through an estrogen response element (ERE)-luciferase reporter gene assay and quantitative polymerase chain reaction analysis of expression of two downstream ERα-regulated endogenous target genes, progesterone receptor (PGR) and trefoil factor 1 (TFF1). Cell uptake FES saturation binding assays and nonlinear regression (one site-total and nonspecific binding) were performed to determine the FES equilibrium dissociation constant, K_D, and the total receptor density, B_max.

Results: Of the mutations tested, Y537S and Y537C demonstrated strong constitutive activity in the absence of estrogen (10- and 5-fold increase in ERE-luciferase reporter gene activation compared to WT ERα, respectively). Constitutive receptor activation of endogenous target gene expression in the absence of estrogen was confirmed for Y537S and Y537C (6-, and 5-fold increase for PGR and 110-, and 25-fold increase for TFF1 compared to WT ERα, respectively). The Y537S and Y537C mutations decreased FES binding affinity despite abundant total receptor density. K_D values were 0.42±0.21 nM and 0.20±0.13 nM vs 0.071±0.026 nM and B_max values were 421±96 and 113±28 vs 85.3±9.0 fmol/mg protein for Y537S and Y537C compared to WT ERα, respectively.

Conclusion: Y537S and Y537C mutations in the ligand binding domain cause robust constitutive receptor functional activity in the absence of estrogen and decrease FES binding affinity. This result has the potential to negatively affect interpretation of FES-positron emission tomography imaging in patients with metastatic ER+ breast cancer, which is currently under investigation. Research Support: Philips Healthcare/Radiological Society of North America Research Seed Grant #RSD1420, University of Wisconsin Paul P. Carbone Cancer Center Young Investigator Award, University of Wisconsin Institute of Clinical and Translational Research KL2 Scholar Award (5KL2TR000428-09, 4KL2TR000428-10), Department of Radiology and University of Wisconsin School of Medicine and Public Health.