Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Abstract

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ER A) gene also oscillates in a circadian fashion. In contrast, ER A-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ER A-positive breast cancer cells do not display circadian oscillation of ER A expression. Our findings suggest that estrogen signaling could be affected not only in ER A-negative breast cancer, but also in ER A-positive breast cancer due to lack of circadian availability of ER A. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.

Figure 1

The mammalian circadian clock. Simplified representation of the cell molecular clock, consisting of an oscillator in which clock gene transcripts and proteins accumulate in a circadian fashion due to a complex feedback loop. The output of this inner oscillator is the circadian transcriptional regulation of many clock-controlled genes (CCGs) directly bound by BMAL1-CLOCK.

Figure 2

ERA-positive human mammary epithelial HME1 cells are endowed with an inner functional clock. Quantitative RT-PCR analysis on cultured HME1 cells at 4-h intervals after entrainment via serum shock shows that the mRNA of the clock genes PER1, PER2, PER3, CRY1, CRY2, RevErbA and BMAL1 accumulates in a circadian fashion, while the mRNA of CLOCK does not show any circadian oscillation.

Figure 3

Entrainment of ERA-negative human mammary epithelial MCF10A cells detects a defective clock. Quantitative RT-PCR at 4 h time points after entrainment shows that in cultured MCF10A cells, only PER1 and PER3 mRNAs have a circadian oscillation comparable with HME1 cells, while the other clock genes display either an imperfect circadian oscillation (CRY1, PER2, RevErbA, BMAL1) or no circadian oscillation (CRY2, CLOCK).

Figure 4

ERA-negative human breast cancer cells show a disrupted inner clock. Quantitative RT-PCR shows that entrainment of the ERA-negative breast cancer cell lines HS578T and MDA-MB-231 cannot induce clock gene mRNA circadian oscillation, as it does in HME1 cells. See the Results section for a more detailed description of the single gene oscillations in each cell line.

Figure 5

ERA-positive human breast cancer cells also display a disrupted inner clock. Quantitative RT-PCR at different time points after entrainment by serum shock shows that the circadian oscillation of clock genes mRNA is lost in ERA-positive T47D and MCF7 breast cancer cells. See the Results section for a more detailed description of the single gene oscillations in each cell line.

Figure 6

ERA mRNA oscillates in a circadian fashion in ERA-positive HME1 cells but not in ERA-positive breast cancer cells. Quantitative RT-PCR at 4 h time points after entrainment by serum shock shows that ERA mRNA oscillates in a circadian fashion, and its accumulation precedes the circadian accumulation of PER2 mRNA in ERA-positive, mammary epithelial HME1 cells. In contrast, ERA-positive T47D and MCF7 breast cancer cells, which do not show PER2 circadian expression, also do not show any ERA mRNA circadian rhythmicity.

Figure 7

The core clock gene PER2 links the circadian oscillator with ERA signaling. PER2 transcription can be induced by estrogen (E2)-ERA signaling; second, PER2 physically interacts with ERA and leads to its degradation; finally, PER2 interacts with RevErbA, RORA and other nuclear receptors (NR) to modulate the transcription of NR-target genes, including BMAL1. Based on references and .