RESEARCH ARTICLE G1/S cell cycle regulators mediate effects of circadian dysregulation on tumor growth and provide targets for timed anticancer treatment 1 2 1 1 3 Yool Lee , Nicholas F. LahensID , Shirley ZhangID , Joseph Bedont , Jeffrey M. Field , 1 Amita SehgalID * 1 Penn Chronobiology, Howard Hughes Medical Institute, Department of Neuroscience, Perelman School of a1111111111 Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, a1111111111 Philadelphia, Pennsylvania, United States of America, 3 Department of Systems Pharmacology and a1111111111 Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, a1111111111 Pennsylvania, United States of America a1111111111 * [email protected] Abstract OPEN ACCESS Citation: Lee Y, Lahens NF, Zhang S, Bedont J, Circadian disruption has multiple pathological consequences, but the underlying mecha- Field JM, Sehgal A (2019) G1/S cell cycle nisms are largely unknown. To address such mechanisms, we subjected transformed cul- regulators mediate effects of circadian tured cells to chronic circadian desynchrony (CCD), mimicking a chronic jet-lag scheme, dysregulation on tumor growth and provide targets and assayed a range of cellular functions. The results indicated a specific circadian clock± for timed anticancer treatment. PLoS Biol 17(4): e3000228. https://doi.org/10.1371/journal. dependent increase in cell proliferation. Transcriptome analysis revealed up-regulation of pbio.3000228 G1/S phase transition genes (myelocytomatosis oncogene cellular homolog [Myc], cyclin Academic Editor: Achim Kramer, Charite - D1/3, chromatin licensing and DNA replication factor 1 [Cdt1]), concomitant with increased UniversitaÈtsmedizin Berlin, GERMANY phosphorylation of the retinoblastoma (RB) protein by cyclin-dependent kinase (CDK) 4/6 Received: September 20, 2018 and increased G1-S progression. Phospho-RB (Ser807/811) was found to oscillate in a cir- cadian fashion and exhibit phase-shifted rhythms in circadian desynchronized cells. Consis- Accepted: March 27, 2019 tent with circadian regulation, a CDK4/6 inhibitor approved for cancer treatment reduced Published: April 30, 2019 growth of cultured cells and mouse tumors in a time-of-day±specific manner. Our study Copyright: © 2019 Lee et al. This is an open access identifies a mechanism that underlies effects of circadian disruption on tumor growth and article distributed under the terms of the Creative underscores the use of treatment timed to endogenous circadian rhythms. Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are Author summary within the paper and its Supporting information files. Circadian misalignment caused by altered sleep±wake cycles, shift work, or frequent jet lag increases susceptibility to several disorders, including cancer. However, the mecha- Funding: This work was supported by the National nisms by which circadian disruption contributes to disease are not well understood, and Institutes of Health (NIH) (R37-NS-048471) and Howard Hughes Medical Institute (HHMI) to A.S. so we addressed this issue by investigating the molecular, cellular, and biochemical conse- The funders had no role in study design, data quences of chronic circadian desynchronization. Our studies using cancer cell or tumor collection and analysis, decision to publish, or tissue models show that chronic circadian desynchronization induces multiple oncogenic preparation of the manuscript. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000228 April 30, 2019 1 / 33 Circadian dysregulation increases tumor growth through a mechanism targeted by anticancer treatment Competing interests: The authors have declared that no competing interests exist. pathways to promote cell proliferation. In particular, chronic circadian desynchronization Abbreviations: AKT, alpha serine/threonine-protein promotes phosphorylation of the retinoblastoma (RB) protein, thereby favoring G1/S kinase serine/threonine kinase; ANXA1, annexin phase cell cycle progression. Consistent with these findings, the antiproliferative activity A1; AP-1, activator protein 1; BAX, B, cell of a selective inhibitor of the enzyme that phosphorylates RB has time-of-day±specific lymphoma 2-associated X; BMAL1, brain and effects on cancer cells and mouse tumors, but this time dependence is abrogated by muscle arnt-like protein-1; BRCA1, breast cancer chronic jet-lag conditions. These data suggest a circadian regulation of G1/S cell cycle pro- type 1 susceptibility protein; CCD, chronic circadian desynchrony; CCNB1, cyclin B1; CCND1, cyclin D1; gression and provide an important molecular rationale for time-of-day±specific treatment CCND3, cyclin D3; CD36, cluster of differentiation of cancer patients, also known as chronotherapy. 36; CDK, cyclin dependent kinase; Cdt1, chromatin licensing and DNA replication factor 1; CLOCK, circadian locomoter output cycles protein kaput; Cry, Cryptochrome; CTL, control; DE, differential expression; DEG, differentially expressed gene; dex, dexamethasone; DHX58, DEXH (Asp-Glu-X- Introduction His) box polypeptide 58; DMEM, Dulbecco's In response to day±night cycles produced by earth's 24-hour rotation around its axis, almost Modified Eagle Medium; E2F, E2 transcription all living organisms have evolved circadian clocks, endogenous timekeeping systems that factor; EGF, epidermal growth factor; EGFR, adapt physiology to daily changes in the environment. In mammals, the circadian timing sys- epidermal growth factor receptor; Em., emission; ERBB2, erbb2 receptor tyrosine kinase 2; ERK, tem consists of a central light-entrained clock in the suprachiasmatic nucleus (SCN) of the extracellular signal-regulated kinase; Ex., excitation; brain and numerous peripheral clocks located in most body organs, all of which are typically EXOC3L2, exocyst complex component 3-like coordinated to constrain sleep and feeding, metabolism, and immune functions to appropriate protein 2; FDA, United States Food and Drug times of the day. The basic timekeeping unit is the cell in that even single neurons and fibro- Administration; FDR, false discovery rate; FGF, blasts harbor a conserved, cell-autonomous circadian clock. At the molecular level, the basic fibroblast growth factor; FGFR, fibroblast growth factor receptor; FITC, fluorescein isothiocyanate; mechanism consists of transcription-translation feedback loops, with the major loop in mam- FUCCI, fluorescence ubiquitination-based cell-cycle mals comprised of the Period (Per) and Cryptochrome (Cry) genes, which are rhythmically indicator; GAPDH, glyceraldehyde 3-phosphate transcribed by circadian locomoter output cycles protein kaput (CLOCK)Ðbrain and muscle dehydrogenase; GC, glucocorticoid; GPCR, G Arnt-like protein-1(BMAL1) transcription factors and repressed by their own protein prod- protein-coupled receptor; GSH, glutathione; GSSG, ucts (PER, CRY). With further fine-tuning at transcriptional, posttranscriptional, and transla- glutathione disulfide; HIPPO, hippo signaling tional levels, the intrinsic molecular oscillator integrates multiple external signals to regulate pathway; HMGA1, high mobility group AT-hook 1; IPA, Ingenuity Pathway Analysis; JAG2, jagged 2; expression of clock-controlled genes, which differ dramatically from tissue to tissue despite JL, jet lag; JUN, jun oncogene; JUND, Jun proto- usage of largely the same core clock genes across the organism [1]. oncogene related gene d; MAFA, Consistent with the adaptive physiological and cellular benefits of the circadian timing sys- musculoaponeurotic fibrosarcoma oncogene A; tem, accumulating evidence indicates that disruption of circadian homeostasis by genetic alter- MAFB, musculoaponeurotic fibrosarcoma ation or irregular lifestyle has pathological consequences [2]. For instance, epidemiological oncogene B; MAPK, mitogen activated protein kinase; MCA, methylcholanthrene; mTOR, studies show that frequent misalignment of circadian rhythms caused by lifestyle factors such mammalian target of rapamycin; MTT, thiazolyl as chronic sleep deprivation, jet lag, or shift work is a potential risk factor for cancer [3]. Even blue tetrazolium bromide; Myc, myelocytomatosis in experimental animal studies, genetic or environmental disruption of circadian rhythms oncogene cellular homolog; NF1, neurofibromin 1; highly increases the incidence or growth rate of various types of tumors, including lung, breast, NFKB, nuclear factor kappa B; NOTCH, notch skin, oral, and prostate cancers [4±6]. The wide variety of tumors affected suggests that circa- signaling pathway; NPAS2, neuronal PAS domain dian disruption alters basic cellular physiology in a fundamental way that increases susceptibil- protein 2; PCSK1N, proprotein convertase subtilisin/kexin type 1 inhibitor; Per, Period; Per2, ity to diseases like cancer; however, the underlying mechanism is not understood. Period2; PFA, paraformaldehyde; PI3K, Increased tumorigenesis and accelerated tumor growth are often linked to a dysregulated phosphatidylinositol 3-kinase/alpha serine/ cell cycle [7,8]. The cell cycle consists of cell growth (G1), DNA replication (S), and cell divi- threonine-protein kinase; PORT, Pipeline of RNA- sion (G2/M) phases. Generally, control of cell proliferation occurs