OR26-2 Phosphorylation of Estrogen Receptor Alpha is Required for Mammary Gland Development and MCF-7 Breast Tumor Response to Estradiol and Tamoxifen

Abstract

Serine-167 (S167) is a major phosphorylation site in estrogen receptor alpha (ERα) and a predictor for endocrine therapy response in breast cancer. We previously showed using stable transfection that mutation of S167 to alanine (S167A) impacted MCF-7 breast cancer cell growth and morphology. Using CRISPR, we evaluated S167A mutation in the endogenous ERα gene in MCF-7 breast cancer cells and xenograft tumors. In vitro, MCF-7 cells with wildtype ERα (WT) exhibited >60% increase in growth after 4 days incubation with 10-9 M 17-β estradiol (E2), and >300% decrease in growth after 4 days incubation with 10-8 M 4-hydroxy-tamoxifen (TAM) as assessed by MTS assay. In marked contrast, S167A cells showed resistance to both E2 growth stimulation and TAM growth arrest. WT cells exhibited >300% increase in migration after 24 hr E2 as assessed by Boyden Chamber assay. S167A cells exhibited >3 fold increase in basal migration compared to WT cells but were insensitive to E2 stimulation of migration. RNAseq was performed on WT and S167A cells incubated with E2 or vehicle (6 hr). Sequencing data was assessed using FastQC and sequenced libraries mapped to the human genome (UCSC hg38) using STAR RNAseq aligner. RNASeq data was normalized using TMM. Differential expression analysis by edgeR revealed significant differences in gene expression between WT and S167A cells. Vehicle-treated S167A cells exhibited 623 differentially regulated genes compared to WT cells. Notably, S167A cells exhibited marked E2 resistance with only 9 genes regulated by E2 in S167A cells compare to 93 genes in WT cells. In tumor xenograft experiments using ovariectomized nude mice, similar to WT tumors the S167A tumors did not grow in the absence of implanted 17 beta-estradiol (0.72 mg/pellet; 60 day release) demonstrating dependency of S167A tumors on E2 for tumor establishment and growth. However, unlike WT tumors that exhibited tumor regression with E2 + TAM (5 mg/pellet; 60 day release) treatment (week 4, P<0.05), S167A tumors were resistant to TAM and exhibited continued growth similar to E2 alone treatment. Serine-212 (S216 in mouse) is a phosphorylation site located between the two zinc fingers in the DNA binding domain and is highly conserved in the nuclear receptor superfamily. To understand the importance of ERα phosphorylation for mammary gland development, ERα knock-in (KI) mice (Esr1S216A) that express the ERα S216A mutation were evaluated. Mammary glands were whole-mounted with carmine-alum from 5 week-old pubertal female ERα wildtype (WT) and KI mice (n=3). KI mice exhibited a significant decrease in ductal elongation (P<0.05), number of terminal end buds (P<0.01) and branching morphogenesis (P<0.01) compared to WT mice. Collectively, these results demonstrate that S216 phosphorylation is critical for mouse mammary gland development and S167 phosphorylation is required for MCF-7 breast tumor response to estradiol and tamoxifen.