Tulane University School of Medicine, Department of Structural and Cellular Biology, 1430 Tulane Avenue SL-49, New Orleans, Louisiana 70112, USA.
Endocrinology. 2012 Sep;153(9):4144-59. doi: 10.1210/en.2011-2001. Epub 2012 Jun 25.
Elevated phosphorylation of estrogen receptor α (ERα) 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 ERα signaling pathway in breast cancer. It is possible that loss of phosphorylation at S118 and/or S167 could disrupt ERα signaling, resulting in aggressive ERα-independent breast cancer cells. To this end, MCF-7 breast cancer cells were stably transfected with an ERα-specific short hairpin RNA that reduced endogenous ERα. The resulting cell line was stably transfected with wild-type ERα (ER-AB cells), or ERα containing serine to alanine mutation at S118 or S167 (S118A cells and S167A cells, respectively). These stable cell lines expressed approximately equivalent ERα compared with parental MCF-7 cells and were evaluated for growth, morphology, migration/invasion, and ERα-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 β-catenin and did not express vimentin, indicating retention of epithelial lineage markers. Expression of ERα-target genes and other genes regulated by ERα signaling or involved in breast cancer were markedly altered in both S118A and S167A cells. In summary, attenuated phosphorylation of ERα 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 ERα target genes, and markedly altered gene expression patterns.
雌激素受体 α(ERα)丝氨酸 118(S118)和 167(S167)的磷酸化水平升高与他莫昔芬辅助治疗的良好预后相关,并且可能作为乳腺癌中功能性 ERα信号通路的替代标志物。S118 和/或 S167 的磷酸化丧失可能破坏 ERα 信号,导致侵袭性的 ERα 非依赖性乳腺癌细胞。为此,用 ERα 特异性短发夹 RNA 稳定转染 MCF-7 乳腺癌细胞,降低内源性 ERα。所得细胞系稳定转染野生型 ERα(ER-AB 细胞)或 ERα 中 S118 或 S167 的丝氨酸突变为丙氨酸(S118A 细胞和 S167A 细胞,分别)。这些稳定细胞系与亲本 MCF-7 细胞相比表达大约相当水平的 ERα,并评估其生长、形态、迁移/侵袭和 ERα 调节的基因表达。S118A 细胞和 S167A 细胞在体外表现出增强的生长和迁移/侵袭。前向和侧向散射流式细胞术显示 S167A 细胞体积较小,S118A 和 S167A 细胞的细胞复杂性较低。S118A 和 S167A 细胞表达细胞质角蛋白和膜定位的β-连环蛋白,不表达波形蛋白,表明保留了上皮谱系标志物。S118A 和 S167A 细胞中 ERα 靶基因和其他受 ERα 信号调节或参与乳腺癌的基因的表达明显改变。总之,ERα 丝氨酸 118 和 167 的磷酸化减弱显著影响 MCF-7 乳腺癌细胞的细胞生理学和行为,导致生长、迁移/侵袭增加,ERα 靶基因表达受损,以及明显改变基因表达模式。