Loss of ERα Phosphorylation at Serine 167 and Serine 212 Alters the ERα transcriptome/cistrome, Impacting Breast Cancer Growth, Tamoxifen Response, Mouse Fertility, Aging and Metabolism

Estrogen receptor alpha (ERα) is a ligand-dependent transcription factor which has multiple phosphorylation sites. Phosphorylation at serine (S) 167 is associated with increased overall survival in breast cancer (BC) patients treated with adjuvant tamoxifen. S212 is a highly conserved phosphorylation site located in the DNA binding domain and is likely critical for DNA binding and gene expression. S to alanine (A) mutations were introduced into the endogenous ERα-gene in MCF7 BC cells by CRISPR-Cas9 to generate ERα phospho knock-in at S167 or S212. Migration, invasion, colony formation, transcriptomes, chromatin interaction ('Cut and Run' assay), and xenograft tumor growth in nude mice were assessed from MCF7 cells expressing either wild-type (WT) ERα, ERα-S167A, or ERα-S212A under conditions of vehicle, 17β-estradiol (E2), or tamoxifen. Corresponding phospho knock-in ERα mutations were created in C57BL6 mice to assess the impact on mouse physiology. Compared to WT cells, S167A cells showed elevated migration/invasion. Unlike WT xenograft tumors in mice, S167A were resistant to tamoxifen growth inhibition, showing no reduction in tumor growth compared to the 65.5% inhibition observed in WT tumors. S212A tumors exhibited significantly impaired growth, reaching only 5.3% of the weight of WT tumors. S167A and S212A cells exhibited very distinct transcriptomes. GSEA indicated enrichment in a tamoxifen-resistant gene set for S167A cells and PTEN pathway gene set for S212A cells. Chromatin binding patterns revealed unique ligand-dependent and ligand-independent binding motifs for both mutant cells. In C57BL6 mice, S216A females exhibited sub-fertility (p<0.001) with 4.31 pups/litter compared to 6.31 in WT. Micro-CT analysis of 3-6 month-old mice revealed that S216A males had reduced adipose tissue mass (33.3%, p=0.003) and increased lean tissue mass (7.5%, p=0.009). S171A females exhibited increased femur bone mineral density (BMD) (3.9%, p=0.037), whereas S216A females exhibited reduced femur BMD (3.3%, p=0.039). In S216A female mice, metabolomic analysis revealed reduced plasma metabolites for key pathways including glycolysis and TCA cycle. S171A and S216A mutant mice exhibited significantly longer lifespans compared to WT, with increase of 29.2% and 30.8% in males and 21.0% and 11.5% in females for S171A and S216A mutations (p<0.05), respectively. Loss of S167 or S212 phosphorylation significantly altered the ERα transcriptome/cistrome affecting cell/tumor growth and tamoxifen response. Corresponding mutations in mice resulted in substantial changes in mouse body composition and physiology, including fertility, metabolism, and aging.

Date of Presentation October 17, 2024