Abstract 1822: Circadian regulation of estrogen receptor alpha protein and phosphorylation in mammary carcinoma cells

Abstract

Circadian regulation of nuclear receptors in peripheral tissues is important in the timing of key physiological processes. However few studies have examined circadian regulation of estrogen receptor alpha (ERa) protein in cancer or normal tissues, and no studies have evaluated ERα phosphorylation during the circadian cycle. In addition to transcription, protein turnover and posttranslational modifications are important regulatory mechanisms in the circadian control of protein function. ERα protein is important in regulation of the peripheral clock proteins Period (PER), Cryptochrome (CRY), CLOCK, and brain and muscle arnt-like protein-1 (BMAL1). Several key ERα phosphorylation sites, serine 118 and serine 167, important in regulating ERα function contain consensus sequences for two kinases, casein kinase 1 and 2 (CK1, CK2), that regulate the peripheral clock proteins levels. The present study examined ERα protein levels and phosphorylation in human and rat mammary carcinoma cells following entrainment by 50% serum shock that results in circadian oscillation of clock genes in vitro. In human MCF-7 breast cancer cells, ERα protein levels exhibited circadian regulation after serum shock with an approx. 60% decrease at 4 hours, recovery to baseline level or above at 16 hours, and a 30% decline from baseline at 24 hours. Estradiol treatment resulted in a longer sustained suppression of ERα protein levels. Rat MAT mammary carcinoma cells exhibited a similar circadian regulation of ERα protein following serum shock. In MCF-7 cells, serine 118 phosphorylation was elevated 2-7 fold 4 hours after serum shock with a subsequent return to baseline levels by 12 hours. Estradiol treatment resulted in a more robust circadian cycle of serine 118 phosphorylation of up to 40 fold with peaks occurring between 4-8 hours and at 16-24 hours. A reverse phosphorylation pattern occurred for serine 167 phosphorylation. Serum shock resulted in a single induction up to 23 fold of serine 167 phosphorylation at 8-12 hours that was suppressed by estradiol. In rat MAT cells, serum shock resulted in circadian oscillation of serine 167 phosphorylation up to 3 fold with peaks at 8 and 20 hours that was similar when cells were treated with estradiol. For both MCF-7 cells and MAT cells, it was noted that peak levels of ERa protein and ERα phosphorylation were out of phase. Estradiol induction of cyclin D1 and c-myc mRNA demonstrated functional ERα signaling following serum shock. These data demonstrate a very significant circadian regulation of ERα protein levels and ERα phosphorylation in mammary carcinoma cells following entrainment by serum shock that likely contribute to time-dependent effects on peripheral clock function and ERα signaling in mammary cancer.[A.Y. and Y.Z. contributed equally to this work.] Citation Format: Alix Youngblood, Yifang Zhang, Murali Anbalagan, Dawei Tian, Brian G. Rowan. Circadian regulation of estrogen receptor alpha protein and phosphorylation in mammary carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1822.