Oncogene (2007) 26, 7916–7920 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION The clock gene Per2 links the circadian system to the estrogen receptor S Gery, RK Virk, K Chumakov, A Yu, HP Koeffler Cedars-Sinai Medical Center, Division of Hematology/Oncology, UCLA School of Medicine, University of California, Los Angeles, CA, USA Circadian rhythms regulate diverse physiological pro- 2007). Consequently in various tissues, circadian cesses including homeostatic functions of steroid hor- rhythms impinge upon many physiological processes mones and their receptors. Estrogen receptor-a (ERa)is and pathological conditions, including cancer (Fu et al., essential for normal mammary gland physiology and is a 2002; Matsuo et al., 2003; Lowrey and Takahashi, 2004; prognostic marker for the treatment of breast cancer. We Ko and Takahashi, 2006). Recent studies suggested that report that Per2, a core clock gene, links the circadian circadian disruption is associated with breast tumor- cycle to the ERa signaling network. Binding of enhances igenesis (Hansen, 2001; Chen et al., 2005). The steroid ERa degradation, while suppression of Per2 levels leads to hormone estrogen is essential for normal mammary ERa stabilization. In turn, Per2 itself is estrogen inducible gland physiology, and is also a potent mammary in these cells, suggesting a feedback mechanism to attenuate mitogen (Sternlicht, 2006; Yager and Davidson, 2006). stimulation by estrogen. In addition, overexpression of Per2 Although the circadian clock has been linked to several in breast cancer cells leads to significant growth inhibition, steroid hormone activities, the molecular mechanisms loss of clonogenic ability and apoptosis. Taken together, underlying the function of core clock genes in mammary these results further support a critical role for peripheral tissue are largely unknown. We hypothesized that circadian regulation in tissue homeostasis and suggest a circadian regulation may be implicated in hormone novel role for clock genes in estrogen receptor-positive homeostasis and hormone-related tumorigenesis in breast cancer. breast epithelia cells. Oncogene (2007) 26, 7916–7920; doi:10.1038/sj.onc.1210585; Estrogen stimulation is thought to be a major factor published online 18 June 2007 contributing to the development of breast cancer. Deregulation of the core clock factor, Per2, has been Keywords: circadian rhythms; ERa; breast cancer; reported in several human malignancies including breast Per2; transcriptional activation; estrogen cancer (Chen et al., 2005). We hypothesized that Per2 function in mammary epithelia cells could be linked to the ERa signaling pathway. ERa regulates transcription of target genes through an interaction with consensus Most physiological processes in mammals are influenced estrogen response elements (EREs). We performed by circadian rhythms. These rhythms are driven by a reporter assays with an ERE-luciferase reporter gene master clock within the hypothalamic suprachiasmatic to test the effect of Per2 on ERa transactivation nuclei (SCN) that synchronizes numerous subsidiary (Figure 1a). The ERa-positive breast cancer cell line, oscillators in peripheral tissues. The circadian clockwork MCF-7, was cotransfected with ERE-luciferase and in both the SCN and the peripheral cells is composed of either Per2 or empty vector. While 17-b estradiol (E2) transcription-translation feedback loops maintained by induced high luciferase activity in the control cells, Per2 a core set of clock genes (Shearman et al., 2000; Reppert expression substantially reduced this activity. Per2 also and Weaver, 2002; Schibler and Sassone-Corsi, 2002; inhibited E2-activated ERE transcription in two addi- Ishida, 2007). Two transcription factors, Clock and tional ERa-positive cancer cell lines, T47D (breast) and Bmal1, activate their targets, Period (Per1, 2 and 3) and Ishikawa (endometrial). In contrast, silencing of Per2 by cryptochrome (Cry1 and Cry2); subsequently, the Per small interfering RNA (siRNA) (as shown in Figure 1d) and Cry proteins interfere with Bmal1:Clock activity in those cells enhanced the ERE reporter activity thereby forming the major negative circadian feedback (Figure 1a). We also examined whether Per2 can loop. The central clock, through neural, hormonal and endogenously suppress ERa-responsive genes (Figures metabolic signals, synchronizes the peripheral oscilla- 1b and c). MCF-7 cells transfected with either Per2 or a tors, which in turn drive the expression of downstream control vector were treated with E2; and the mRNA clock-controlled genes in a tissue-specific manner levels of the known ERa targets, pS2, cyclin D1 and (Panda et al., 2002; Storch et al., 2002; Miller et al., CCN1 were measured by real-time PCR (Figure 1b). While expression of Per2 alone had little effect on Correspondence: S Gery, Cedars-Sinai Medical Center, Davis Building expression of those genes, it strongly inhibited E2-mediated 5066, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA. E-mail: [email protected] induction of pS2, and further reduced the levels of Received 26March 2007; revised 8 May 2007; accepted 9 May 2007; cyclin D1 and CCN1 below basal level. Moreover, published online 18 June 2007 Per2 siRNA enhanced E2-activation of the ERa target Per2 links the circadian system to ER S Gery et al 7917 a b 300 c EV Per2 siCon siPer2 2 pS2 EV 200 Per2 Cyclin D1 1 100 0 (% of EV+E2) CCN1 200 Luciferase activity 0 Cyclin D1 −−+ − + ++−−+ − + E2 MCF-7 T47D Ishikawa 100 β-actin 4 siCon E2 −−++−−++ siPer2 0 Relative expression 200 d 2 CCN1 siCon siPer2 siCon siPer2 100 (% of siCon+E2) Per2 Luciferase activity 0 −−+ − + ++−−+ − + E2 0 β MCF-7 T47D Ishikawa E2 − + − + − + − + -actin EV 293T MCF-7 Per2 siCon siPer2 Figure 1 Per2 suppresses ERa transcriptional activation. (a) MCF-7, T47D (breast) and Ishikawa (endometrial) cancer cell lines were cotransfected with ERE-luciferase construct and either empty vector (EV) or Per2 (Per2, upper panel) or with either a control siRNA (siCon) or Per2 siRNA (siPer2, bottom panel). Luciferase activity was measured either with or without treatment of cells with E2 (1 mM, 16h). Results represent the percentages of luciferase activity with either E2-treated EV-transfected cells (upper panel) or E2- treated siCon-transfected cells (bottom panel) set to 100%. Shown are the means7s.d. of triplicate samples. (b and c) MCF-7 cells transfected with either empty vector or Per2 were selected with G418 for 5 days. Surviving cells were treated with E2 (1 mM, 16h) and harvested for RNA and whole-cell protein. In addition, MCF-7 cells were transfected with either siCon or siPer2. Two days later, cells were treated with E2 (1 mM, 16h) and harvested for RNA and whole-cell protein. ( b) Real-time PCR analysis of the indicated genes. (c) Western blot analysis of the indicated proteins. (d) 293T and MCF-7 cells were transfected with either siCon or siPer2. Nuclear lysates were analysed for Per2 expression by immunoblotting. Cells culture, constructs, real-time PCR conditions and sources of antibodies and regents have been described in the Supplementary Information. genes (Figure 1b). Western blot analysis showed a prevent nuclear localization of ERa.Colocalizationof parallel effect of Per2 on the protein levels of cyclin D1 Per2 and ERa was noted in both cell lines. Notably, Per2- and CCN1 (Figure 1c). These results suggest that Per2 expressing MCF-7 cells exhibited changes in morphology could be involved in breast cancer prevention by inhibit- indicative of apoptosis, such as cell rounding and ing E2-induced proliferation. Recently, Per2 was also detachment, while the 293T cells had normal morphology. suggested to play a role in normal mammary cell differen- ERa is a short-lived protein and binding of E2 further tiation (Metz et al., 2006). accelerates its degradation (Reid et al., 2002). While In the absence of ligand, ERa is sequestered in the Per2 expression had no effect on ERa mRNA levels cytoplasm in an inhibitory protein complex. Upon (Figure 2d), it markedly downregulated ERa protein binding to E2, ERa undergoes conformational changes levels in MCF-7 cells (Figure 2e). PS-341, a specific facilitating cofactor binding and nuclear localization proteasome inhibitor, blocked Per2-stimulated down- (McDonnell and Norris, 2002). Immunoprecipitation regulation of ERa (Figure 2f) showing that Per2- experiments showed that Per2 associates with ERa in mediated ERa degradation is through the proteasome 293T and MCF-7 cells, and E2 stimulation enhances pathway. Moreover, suppression of endogenously the interaction (Figure 2a). Moreover, glutathione expressed Per2 by siRNA led to stabilization of ERa S-transferase (GST) pull-down assays, with an in vitro- (Figure 2g), suggesting that Per2 is necessary for translated Per2 and a GST-ERa fusion protein, efficient proteasome-induced degradation of ERa.A demonstrated direct binding of these proteins (Figure 2b). recent study showed that several nuclear receptors are As Per2 shuttles between the nucleus and the cytoplasm clock-controlled genes (Yang et al., 2006). Although (Yagita et al., 2002), it could change the subcellular ERa does not exhibit a circadian expression pattern in distribution of ERa. To test this possibility, 293T cells normal cells, it is downregulated in Clock mutant mice cotransfected with ERa and V5-tagged mPer2, and (Miller et al., 2007), suggesting that core circadian MCF-7 cells transfected with V5-tagged mPer2 were components have an important