Stable Inhibition of Specific Estrogen Receptor α (ERα) Phosphorylation Confers Increased Growth, Migration/Invasion, and Disruption of Estradiol Signaling in MCF-7 Breast Cancer Cells

Elevated phosphorylation of estrogen receptor alpha (ER alpha) at serines 118 (S118) and 167 (S167) is associated with favorable outcome for tamoxifen adjuvant therapy and may serve as surrogate markers for a functional ER alpha signaling pathway in breast cancer. It is possible that loss of phosphorylation at S118 and/or S167 could disrupt ER alpha signaling, resulting in aggressive ER alpha-independent breast cancer cells. To this end, MCF-7 breast cancer cells were stably transfected with an ER alpha-specific short hairpin RNA that reduced endogenous ER alpha. The resulting cell line was stably transfected with wild-type ER alpha (ER-AB cells), or ER alpha containing serine to alanine mutation at S118 or S167 (S118A cells and S167A cells, respectively). These stable cell lines expressed approximately equivalent ER alpha compared with parental MCF-7 cells and were evaluated for growth, morphology, migration/invasion, and ER alpha-regulated gene expression. S118A cells and S167A cells exhibited increased growth and migration/invasion in vitro. Forward- and side-scatter flow cytometry revealed that S167A cells were smaller in size, and both S118A and S167A cells exhibited less cellular complexity. S118A and S167A cells expressed pancytokeratin and membrane localization of beta-catenin and did not express vimentin, indicating retention of epithelial lineage markers. Expression of ER alpha-target genes and other genes regulated by ER alpha signaling or involved in breast cancer were markedly altered in both S118A and S167A cells. In summary, attenuated phosphorylation of ER alpha at S118 and S167 significantly affected cellular physiology and behavior in MCF-7 breast cancer cells, resulting in increased growth, migration/invasion, compromised expression of ER alpha-target genes, and markedly altered gene expression patterns. (Endocrinology 153: 4144-4159, 2012)