A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

https://doi.org/10.1016/j.bbrc.2015.09.103Get rights and content

Highlights

  • Cryptochrome inhibitor (KS15) has anti-tumor activity to human breast cancer cells.

  • KS15 induces differential changes in cell cycle regulators and pro-apoptotic genes.

  • KS15 inhibits MCF-7 cell growth and enhances susceptibility to anti-tumor drugs.

Abstract

Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes, were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer.

Introduction

The circadian rhythm plays a fundamental role in the regulation of physiology and behavior in almost all organisms on Earth. In mammals, several lines of evidence indicate that nearly all biological processes, from cellular proliferation to higher brain functions, are influenced by circadian rhythms [1], [2]. Consequently, disruption of circadian rhythm is associated with a variety of diseases [3], [4], and the regulation of circadian rhythm is a clinically important pathway.

Maintenance of the circadian rhythm is largely dependent on interlocking networks among the core clock genes, called the molecular circadian clockwork (MCC) [5]. The MCC, which comprises negative and positive feedback loops, generates daily oscillatory expression of downstream genes, called clock-controlled genes (CCGs) [6], [7]. The positive limb of the MCC consists of two core components, Circadian Locomotor Output Cycle Kaput (CLOCK) and Brain-Muscle-Arnt-Like protein 1 (BMAL1). CLOCK and BMAL1 interact with each other, and the resultant heterodimer acts as a transcriptional activator by recognizing E-box elements in promoters of downstream genes. Period (PER) and Cryptochrome (CRY), which are directly regulated by the CLOCK:BMAL1 heterodimer, translocate into the nucleus after translation and form a negative complex that suppresses CLOCK:BMAL1-mediated transcription, thereby completing the core feedback loop [8], [9]. A secondary loop is composed of nuclear receptors, including RAR-related Orphan Receptors (RORs) and REV-ERBα/β, which stabilize the normal cycling of the core clock loop. REV-ERBα/β are transcriptional repressors that compete with the RORs to regulate periodic BMAL1 gene transcription. Moreover, the RORs and REV-ERBα/β are under the control of CLOCK:BMAL1-mediated transactivation, connecting the secondary loop with the core loop of the circadian molecular clock [10].

Recently discovered synthetic modulators of the MCC have emerged as useful tools for investigating the regulatory mechanisms and clinical implications of clock genes [11]. For example, synthetic antagonists [12] and agonists [13] of REV-ERBα/β were recently identified and tested in various disease models, including metabolic syndrome [13]. In addition, a very recently developed small-molecule activator of CRY1/2 exerted an inhibitory effect on gluconeogenesis in hepatocytes, which is important for regulation of blood glucose levels in diabetes [14]. In our previous study, we discovered a novel inhibitor of CRY, KS15, which can activate CLOCK:BMAL1-evoked E-box-mediated transcription [15].

Breast cancer, a common cancer in women in most developed countries, is closely associated with disturbances of the circadian rhythm [16], [17]. Moreover, many risk factors for breast cancer are related to the status of core clock genes [18], [19]. Importantly, essential cell-cycle regulators and tumor suppressors, members of the CCGs and circadian control genes could lead to appropriate coupling of cell proliferation with key tissue functions in vivo [20], [21]. CRY is involved in DNA-damage checkpoint controls [22] and cell-cycle progression [20], [23]. Notably, CRY1/2 mutation exerts a protective effect against cancer-prone phenotypes in response to genotoxic stress [23], [24], [25]. Based on the connection between CRY and cell proliferation, researchers have been investigating the relationship between CRY and the risk of breast cancer [19], [26]. Notably, knockdown of CRY2 in a human breast cancer cell line exerted an adverse effect on regulation of DNA-damage repair and the maintenance of genomic stability [27]. Taken together, these findings suggest that functional modulation of CRY represents a promising novel strategy for treating breast cancer. In this present study, we investigated whether pharmacological inhibition of CRY could exert anti-tumor activity in human breast cancer cells.

Section snippets

Materials

KS15 and KS25 [15] were prepared as 0.02 M stock solutions in 100% dimethyl sulfoxide (DMSO), stored at −70 °C, and diluted with medium before each experiment. Throughout the study, the final DMSO concentration did not exceed 0.2%. DMSO and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Before each usage, MTT was dissolved in complete culture media (0.5 mg/ml). Doxorubicin (DOX) and tamoxifen (TAM) were also purchased

KS15 modulates expression profiles of circadian clock genes as well as various cell-cycle regulators and pro-apoptotic genes

We previously showed that KS15 can increase the activity of the CLOCK:BMAL1 heterodimer, thereby increasing the output of the MCC [15]. Therefore, we hypothesized that KS15 treatment could also evoke the same changes in clock gene expression in human breast cancer cells. MCF-7 cells are a well-defined in vitro model for breast cancer, and several studies used these cells to investigate growth inhibition and pro-apoptotic activity induced by modulations of clock genes [30], [31]. Based on these

Discussion

Development of novel synthetic modulators of the circadian clock, as well as application of these ‘clock modulators’ to various disease models, are emerging fields in translational biomedical science [11]. However, despite its potential clinical importance, studies aimed at discovering the anti-tumor activity of synthetic clock modulators have begun only recently. In this study, we demonstrated that KS15, a recently developed specific inhibitor of CRY [15], can inhibit proliferation and

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

This study was supported by grants of the Korea Healthcare Technology R&D project from the Ministry for Health & Welfare Affairs, Republic of Korea (A121549-1201-0000100) and the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning (NRF-2013M3C7A1056731). This work was also supported by the DGIST MIREBraiN Program of the Ministry of Science, ICT, & Future Planning.

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