CLOCK

Gene Summary

Gene:CLOCK; clock circadian regulator
Aliases: KAT13D, bHLHe8
Location:4q12
Summary:The protein encoded by this gene plays a central role in the regulation of circadian rhythms. The protein encodes a transcription factor of the basic helix-loop-helix (bHLH) family and contains DNA binding histone acetyltransferase activity. The encoded protein forms a heterodimer with ARNTL (BMAL1) that binds E-box enhancer elements upstream of Period (PER1, PER2, PER3) and Cryptochrome (CRY1, CRY2) genes and activates transcription of these genes. PER and CRY proteins heterodimerize and repress their own transcription by interacting in a feedback loop with CLOCK/ARNTL complexes. Polymorphisms in this gene may be associated with behavioral changes in certain populations and with obesity and metabolic syndrome. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2014]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:circadian locomoter output cycles protein kaput
Source:NCBIAccessed: 16 March, 2017

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 16 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Messenger RNA
  • Nuclear Proteins
  • Base Sequence
  • Sjogren's Syndrome
  • Case-Control Studies
  • Neoplastic Cell Transformation
  • Transcription
  • Cell Cycle Proteins
  • DNA Methylation
  • Stomach Cancer
  • Biological Clocks
  • Gene Expression Profiling
  • Cryptochromes
  • Breast Cancer
  • Period Circadian Proteins
  • Transcription Factors
  • Gene Expression Regulation
  • Genetic Predisposition
  • CLOCK Proteins
  • AKT1
  • Cell Proliferation
  • Aging
  • Telomerase
  • Tandem Repeat Sequences
  • ARNTL Transcription Factors
  • Colorectal Cancer
  • Virus Replication
  • Apoptosis
  • Cell Aging
  • Risk Factors
  • Chromosome 4
  • Cancer Gene Expression Regulation
  • Biomarkers, Tumor
  • CLOCK
  • beta Catenin
  • Young Adult
  • RTPCR
  • Cell Cycle
  • Melatonin
  • Circadian Rhythm
  • Circadian Clocks
Tag cloud generated 16 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (3)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CLOCK (cancer-related)

Chang L, Li L, Li W, et al.
Research on radiotherapy at different times of the day for inoperable cervical cancer.
Int J Clin Pharmacol Ther. 2016; 54(11):856-864 [PubMed] Related Publications
PURPOSE: To investigate the radiation effects and acute damage in inoperable cervical cancer patients irradiated at different times as well as the underlying mechanisms.
METHODS: 67 patients were randomized to a morning group (MG, 9:00 - 11:00 AM) and an evening group (EG, 9:00 - 11:00 PM) and both received external beam radiotherapy (RT) (50 Gy in 25 fractions) at different times. Brachytherapy (36 - 42 Gy in 6 - 7 fractions) was also performed to enhance the radiation response twice every week in all patients at the same time. Clinical therapeutic effects and acute toxicities were evaluated after RT. Flow cytometry was analyzed before and after RT.
RESULTS: Patients' response to radiation was similar in the two groups. Incidences of overall and high-grade (III - IV) diarrhea in the MG vs. the EG were 75.0% vs. 57.6% and 12.5% vs. 6.1%, respectively. The incidence of severe hematological toxicity in the EG was significantly increased compared to the MG group. Cell apoptosis in the EG was significantly higher at 9:00 - 11:00 PM than that at 9:00 - 11:00 AM after RT. No significant differences were found in Gap Phase 0/Gap Phase 1 (G0/G1), Gap Phase 2/Metaphase Phase (G2/M), and Synthesis Phase (S) phase between different times and groups, nor were expressions of Per1, Per2, and Clock. But expressions of Per1, Per2, and Clock were significantly negative with G2/M phase and positively correlated with cell apoptosis.
CONCLUSION: RT at different time intervals results in similar efficacy. However, RT in the morning reduces severe hematological toxicity. Radiation responses may be associated with circadian genes by influence of cell cycles and apoptosis.
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Poletini MO, de Assis LV, Moraes MN, Castrucci AM
Estradiol differently affects melanin synthesis of malignant and normal melanocytes: a relationship with clock and clock-controlled genes.
Mol Cell Biochem. 2016; 421(1-2):29-39 [PubMed] Related Publications
Melanin production within melanocytes is regulated, among others, by estradiol, whose effects on melanogenesis are still not completely elucidated. Here we show that although 10(-7) M 17β-estradiol (E2) increased tyrosinase mRNA levels in B16-F10 malignant melanocytes, there was a transient decrease and abolishment of the temporal variation of melanin content. Both parameters were much higher in the malignant than in normal Melan-a cells. Considering that silencing clock machinery in human melanocytes increases melanogenesis, we investigated clock gene expression in those cell lines. Except for Melan-a Bmal1 and B16-F10 Per2 expression of control cells, Per1, Per2, and Bmal1 expression increased independently of cell type or E2 treatment after 24 h. However, melanoma cells showed a marked increase in Per1 and Bma11 expression in response to E2 at the same time points, what may rule out E2 as a synchronizer agent since the expression of those genes were not in antiphase. Next, we investigated the expression of Xpa, a clock-controlled gene, which in Melan-a cells, peaked at 18 h, and E2 treatment shifted this peak to 24 h, whereas B16-F10 Xpa expression peaked at 24 h in both control and E2 group, and it was higher compared to Melan-a cells in both groups. Therefore, malignant and normal melanocytes display profound differences on core elements of the local clock, and how they respond to E2, what is most probably determinant of the differences seen on melanin synthesis and Tyrosinase and Xpa expression. Understanding these processes at the molecular level could bring new strategies to treat melanoma.

Huisman SA, Ahmadi AR, IJzermans JN, et al.
Disruption of clock gene expression in human colorectal liver metastases.
Tumour Biol. 2016; 37(10):13973-13981 [PubMed] Free Access to Full Article Related Publications
The circadian timing system controls about 40 % of the transcriptome and is important in the regulation of a wide variety of biological processes including metabolic and proliferative functions. Disruption of the circadian clock could have significant effect on human health and has an important role in the development of cancer. Here, we compared the expression levels of core clock genes in primary colorectal cancer (CRC), colorectal liver metastases (CRLM), and liver tissue within the same patient. Surgical specimens of 15 untreated patients with primary CRC and metachronous CRLM were studied. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression of 10 clock genes: CLOCK, BMAL1, PER1, PER2, PER3, CRY1, CRY2, CSNK1E, TIM, TIPIN, and 2 clock-controlled genes: Cyclin-D1, and WEE1. Expression levels of 7 core clock genes were downregulated in CRLM: CLOCK (p = 0.006), BMAL1 (p = 0.003), PER1 (p = 0.003), PER2 (p = 0.002), PER3 (p < 0.001), CRY1 (p = 0.002), and CRY2 (p < 0.001). In CRC, 5 genes were downregulated: BMAL1 (p = 0.02), PER1 (p = 0.004), PER2 (p = 0.008), PER3 (p < 0.001), and CRY2 (p < 0.001). CSNK1E was upregulated in CRC (p = 0.02). Cyclin-D1 and WEE1 were both downregulated in CRLM and CRC. Related to clinicopathological factors, a significant correlation was found between low expression of CRY1 and female gender, and low PER3 expression and the number of CRLM. Our data demonstrate that the core clock is disrupted in CRLM and CRC tissue from the same patient. This disruption may be linked to altered cell-cycle dynamics and carcinogenesis.

Reszka E, Przybek M
Circadian Genes in Breast Cancer.
Adv Clin Chem. 2016; 75:53-70 [PubMed] Related Publications
Exploring the putative impact of circadian rhythms is a relatively novel approach to illuminating hormone-related female breast cancer etiology and prognosis. One of several proposed mechanisms underlying breast cancer risk among individuals exposed to light at night involves circadian gene alterations. Although in vitro and animal studies indicate a key role of circadian genes in breast tumor suppression, there is a paucity of data on the role of circadian genes in human breast cancer. This review summarizes recent findings of circadian gene expression and DNA methylation profile from human breast cancer studies in relation to hormonal status, clinicopathological features of tumors, and exposure to night shift work. The major findings from human studies indicate that expression of circadian genes is deregulated in breast cancer. Breast cancer etiology and prognosis-associated PERs, CRYs, CLOCK downregulation, and TIMELESS upregulation may be related to relevant gene methylation in tumor tissue. Alterations and desynchronization of molecular clock machinery found on genetic and epigenetic level were observed in more aggressive breast cancer tumors and those lacking estrogen receptors.

Gutierrez D, Arbesman J
Circadian Dysrhythmias, Physiological Aberrations, and the Link to Skin Cancer.
Int J Mol Sci. 2016; 17(5) [PubMed] Free Access to Full Article Related Publications
Circadian rhythms are core regulators of a variety of mammalian physiologic processes and oscillate in a 24-h pattern. Many peripheral organs possess endogenous rhythmicity that is then modulated by a master clock; the skin is one of these peripheral organs. The dysregulation of rhythms is associated with decreased ability to ameliorate cellular stressors at a local and global level, which then increases the propensity for the development of neoplastic growths. In this article, we review the implications of altered circadian rhythms on DNA repair as well as modified gene expression of core clock proteins with particular focus on skin models. These findings are then correlated with epidemiologic data regarding skin cancer to showcase the effects of circadian disruption on this phenomenon.

Chen L, Zhao J, Tang Q, et al.
PFKFB3 Control of Cancer Growth by Responding to Circadian Clock Outputs.
Sci Rep. 2016; 6:24324 [PubMed] Free Access to Full Article Related Publications
Circadian clock dysregulation promotes cancer growth. Here we show that PFKFB3, the gene that encodes for inducible 6-phosphofructo-2-kinase as an essential supporting enzyme of cancer cell survival through stimulating glycolysis, mediates circadian control of carcinogenesis. In patients with tongue cancers, PFKFB3 expression in both cancers and its surrounding tissues was increased significantly compared with that in the control, and was accompanied with dys-regulated expression of core circadian genes. In the in vitro systems, SCC9 tongue cancer cells displayed rhythmic expression of PFKFB3 and CLOCK that was distinct from control KC cells. Furthermore, PFKFB3 expression in SCC9 cells was stimulated by CLOCK through binding and enhancing the transcription activity of PFKFB3 promoter. Inhibition of PFKFB3 at zeitgeber time 7 (ZT7), but not at ZT19 caused significant decreases in lactate production and in cell proliferation. Consistently, PFKFB3 inhibition in mice at circadian time (CT) 7, but not CT19 significantly reduced the growth of implanted neoplasms. Taken together, these findings demonstrate PFKFB3 as a mediator of circadian control of cancer growth, thereby highlighting the importance of time-based PFKFB3 inhibition in cancer treatment.

Puram RV, Kowalczyk MS, de Boer CG, et al.
Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML.
Cell. 2016; 165(2):303-16 [PubMed] Article available free on PMC after 07/04/2017 Related Publications
Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.

Wang Q, Ao Y, Yang K, et al.
Circadian clock gene Per2 plays an important role in cell proliferation, apoptosis and cell cycle progression in human oral squamous cell carcinoma.
Oncol Rep. 2016; 35(6):3387-94 [PubMed] Related Publications
Previous studies have shown that the aberrant expression of period circadian clock 2 (Per2) is closely related to the occurrence and development of cancers, but the specific mechanism remains unclear. In the present study, we used shRNA to downregulate Per2 in oral squamous cell carcinoma (OSCC) Tca8113 cells, and then detected the alterations in cell cycle, cell proliferation and apoptosis by flow cytometric analysis and mRNA expression alterations in all the important genes in the cyclin/cyclin-dependent protein kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) cell cycle network by RT-qPCR. We found that in the Tca8113 cells, after Per2 downregulation, the mRNA expression levels of cyclin A2, B1 and D1, CDK4, CDK6 and E2F1 were significantly increased (P<0.05), the mRNA expression levels of p53, p16 and p21 were significantly decreased (P<0.05), cell proliferation was significantly higher (P<0.05), apoptosis was significantly lower (P<0.05) and the number of cells in the G1/G0 phase was significantly decreased (P<0.05). The present study proves that in OSCC, clock gene Per2 plays an important role in cell cycle progression and the balance of cell proliferation and apoptosis by regulation of the cyclin/CDK/CKI cell cycle network. Further research on Per2 may provide a new effective molecular target for cancer treatments.

Gutiérrez-Monreal MA, Treviño V, Moreno-Cuevas JE, Scott SP
Identification of circadian-related gene expression profiles in entrained breast cancer cell lines.
Chronobiol Int. 2016; 33(4):392-405 [PubMed] Related Publications
Cancer cells have broken circadian clocks when compared to their normal tissue counterparts. Moreover, it has been shown in breast cancer that disruption of common circadian oscillations is associated with a more negative prognosis. Numerous studies, focused on canonical circadian genes in breast cancer cell lines, have suggested that there are no mRNA circadian-like oscillations. Nevertheless, cancer cell lines have not been extensively characterized and it is unknown to what extent the circadian oscillations are disrupted. We have chosen representative non-cancerous and cancerous breast cell lines (MCF-10A, MCF-7, ZR-75-30, MDA-MB-231 and HCC-1954) in order to determine the degree to which the circadian clock is damaged. We used serum shock to synchronize the circadian clocks in culture. Our aim was to initially observe the time course of gene expression using cDNA microarrays in the non-cancerous MCF-10A and the cancerous MCF-7 cells for screening and then to characterize specific genes in other cell lines. We used a cosine function to select highly correlated profiles. Some of the identified genes were validated by quantitative polymerase chain reaction (qPCR) and further evaluated in the other breast cancer cell lines. Interestingly, we observed that breast cancer and non-cancerous cultured cells are able to generate specific circadian expression profiles in response to the serum shock. The rhythmic genes, suggested via microarray and measured in each particular subtype, suggest that each breast cancer cell type responds differently to the circadian synchronization. Future results could identify circadian-like genes that are altered in breast cancer and non-cancerous cells, which can be used to propose novel treatments. Breast cell lines are potential models for in vitro studies of circadian clocks and clock-controlled pathways.

Holick MF
Biological Effects of Sunlight, Ultraviolet Radiation, Visible Light, Infrared Radiation and Vitamin D for Health.
Anticancer Res. 2016; 36(3):1345-56 [PubMed] Related Publications
Humans evolved in sunlight and had depended on sunlight for its life giving properties that was appreciated by our early ancestors. However, for more than 40 years the lay press and various medical and dermatology associations have denounced sun exposure because of its association with increased risk for skin cancer. The goal of this review is to put into perspective the many health benefits that have been associated with exposure to sunlight, ultraviolet A (UVA) ultraviolet B (UVB), visible and infrared radiation.

Repouskou A, Prombona A
c-MYC targets the central oscillator gene Per1 and is regulated by the circadian clock at the post-transcriptional level.
Biochim Biophys Acta. 2016; 1859(4):541-52 [PubMed] Related Publications
Cell proliferation in mammals follows a circadian rhythm while disruption of clock gene expression has been linked to tumorigenesis. Expression of the c-Myc oncogene is frequently deregulated in tumors, facilitating aberrant cell proliferation. c-MYC protein levels display circadian rhythmicity, which is compatible with an in vitro repressive role of the clock-activating complex BMAL1/CLOCK on its promoter. In this report, we provide evidence for the in vivo binding of the core circadian factor BMAL1 on the human c-Myc promoter. In addition, analysis of protein synthesis and degradation rates, as well as post-translational acetylation, demonstrate that the clock tightly controls cellular MYC levels. The oncoprotein itself is a transcription factor that by responding to mitogenic signals regulates the expression of several hundred genes. c-MYC-driven transcription is generally exerted upon dimerization with MAX and binding to E-box elements, a sequence that is also recognized by the circadian heterodimer. Our reporter assays reveal that the MYC/MAX dimer cannot affect transcription of the circadian gene Per1. However, when overexpressed, c-MYC is able to repress Per1 transactivation by BMAL1/CLOCK via targeting selective E-box sequences. Importantly, upon serum stimulation, MYC was detected in BMAL1 protein complexes. Together, these data demonstrate a novel interaction between MYC and circadian transactivators resulting in reduced clock-driven transcription. Perturbation of Per1 expression by MYC constitutes a plausible alternative explanation for the deregulated expression of clock genes observed in many types of cancer.

Okazaki F, Matsunaga N, Okazaki H, et al.
Circadian Clock in a Mouse Colon Tumor Regulates Intracellular Iron Levels to Promote Tumor Progression.
J Biol Chem. 2016; 291(13):7017-28 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found thatIrp2mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(Δ19) tumors expressing the clock-mutant protein (CLOCK(Δ19)) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK(Δ19)expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.

Jiang W, Zhao S, Jiang X, et al.
The circadian clock gene Bmal1 acts as a potential anti-oncogene in pancreatic cancer by activating the p53 tumor suppressor pathway.
Cancer Lett. 2016; 371(2):314-25 [PubMed] Related Publications
Disruption of the circadian clock has been shown to be associated with tumor development. This study aimed to investigate the role of the core circadian gene Bmal1 in pancreatic cancer (PC). We first found that the levels of Bmal1 were downregulated in PC samples and were closely correlated with the clinicopathological features of patients. To dissect the underlying mechanism, we performed a RNA-seq assay followed by systematic gene function and pathway enrichment analyses. We detected an anti-apoptotic and pro-proliferative transcriptome profile after Bmal1 knockdown in PC cells. Further in vitro and in vivo studies confirmed that Bmal1 overexpression significantly inhibited cell proliferation and invasion and induced G2/M cell cycle arrest, whereas Bmal1 knockdown promoted PC growth, as demonstrated in Bmal1-manipulated AsPC-1 and BxPC-3 cell lines. Our mechanistic studies indicated that Bmal1 could directly bind to the p53 gene promoter and thereby transcriptionally activate the downstream tumor suppressor pathway in a p53-dependent manner. In sum, our findings suggest that Bmal1 acts as an anti-oncogene in PC and represents a potential biomarker for its diagnosis.

Yang MY, Lin PM, Hsiao HH, et al.
Up-regulation of PER3 Expression Is Correlated with Better Clinical Outcome in Acute Leukemia.
Anticancer Res. 2015; 35(12):6615-22 [PubMed] Related Publications
BACKGROUND: Altered expression of circadian clock genes has been linked to various types of cancer. This study aimed to investigate whether these genes are also altered in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL).
MATERIALS AND METHODS: The expression profiles of nine circadian clock genes of peripheral blood (PB) leukocytes from patients with newly-diagnosed AML (n=41), ALL (n=23) and healthy individuals (n=51) were investigated.
RESULTS: In AML, the expression of period 1 (PER1), period 2 (PER2), period 3 (PER3), cryptochrome 1 (CRY1), cryptochrome 2 (CRY2), brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1), and timeless (TIM) was significantly down-regulated, while that of CK1ε was significantly up-regulated. In ALL, the expression of PER3 and CRY1 was significantly down-regulated, whereas those of CK1ε and TIM were significantly up-regulated. Recovery of PER3 expression was observed in both patients with AML and those with ALL who achieved remission but not in patients who relapsed after treatment.
CONCLUSION: Circadian clock genes are altered in patients with acute leukemia and up-regulation of PER3 is correlated with a better clinical outcome.

Lu H, Chu Q, Xie G, et al.
Circadian gene expression predicts patient response to neoadjuvant chemoradiation therapy for rectal cancer.
Int J Clin Exp Pathol. 2015; 8(9):10985-94 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
Preoperative neoadjuvant chemoradiation therapy may be useful in patients with operable rectal cancer, but treatment responses are variable. We examined whether expression levels of circadian clock genes could be used as biomarkers to predict treatment response. We retrospectively analyzed clinical data from 250 patients with rectal cancer, treated with neoadjuvant chemoradiation therapy in a single institute between 2011 and 2013. Gene expression analysis (RT-PCR) was performed in tissue samples from 20 patients showing pathological complete regression (pCR) and 20 showing non-pCR. The genes analyzed included six core clock genes (Clock, Per1, Per2, Cry1, Cry2 and Bmal1) and three downstream target genes (Wee1, Chk2 and c-Myc). Patient responses were analyzed through contrast-enhanced pelvic MRI and endorectal ultrasound, and verified by histological assessment. pCR was defined histologically as an absence of tumor cells. Among the 250 included patients, 70.8% showed regression of tumor size, and 18% showed pCR. Clock, Cry2 and Per2 expressions were significantly higher in the pCR group than in the non-pCR group (P<0.05), whereas Per1, Cry1 and Bmal1 expressions did not differ significantly between groups. Among the downstream genes involved in cell cycle regulation, c-Myc showed significantly higher expression in the pCR group (P<0.05), whereas Wee1 and Chk2 expression did not differ significantly between groups. Circadian genes are potential biomarkers for predicting whether a patient with rectal cancer would benefit from neoadjuvant chemoradiation therapy.

Alexandrov LB, Jones PH, Wedge DC, et al.
Clock-like mutational processes in human somatic cells.
Nat Genet. 2015; 47(12):1402-7 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
During the course of a lifetime, somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals, and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated, indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This study provides the first survey of clock-like mutational processes operating in human somatic cells.

Gutiérrez-Monreal MA, Villela L, Baltazar S, et al.
A PER3 polymorphism is associated with better overall survival in diffuse large B-cell lymphoma in Mexican population.
Cancer Biomark. 2015; 15(5):699-705 [PubMed] Related Publications
BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma. Presently, one of the most important clinical predictors of survival in DLBCL patients is the International Prognostic Index (IPI). Circadian rhythms are the approximate 24 hour biological rhythms with more than 10 genes making up the molecular clock.
OBJECTIVE: Determine if functional single nucleotide polymorphism in circadian genes may contribute to survival status in patients diagnosed with diffuse large B-cell lymphoma.
METHODS: Sixteen high-risk non-synonymous polymorphisms in circadian genes (CLOCK, CRY2, CSNK1E, CSNK2A1, NPAS2, PER1, PER2, PER3, PPP2CA, and TIM) were genotyped by screening PCR. Results were visualized by agarose gel electrophoresis and confirmed by two-direction sequencing. Clinical variables were compared between mutated and non-mutated groups. LogRank survival analysis and Kaplan-Meier method were used to calculate the overall survival.
RESULTS: PER3 rs10462020 variant showed significant difference in overall survival between patients containing mutated genotypes and those with non-mutated genotypes (p = 0.047). LDH levels (p = 0.021) and IPI score (p < 0.001) also showed differences in overall survival. No clinical differences were observed in mutated vs. non-mutated patients.
CONCLUSIONS: This work suggests a role of PER3 rs10462020 in predicting a prognosis in DLBCL overall survival of patients.

Ercolani L, Ferrari A, De Mei C, et al.
Circadian clock: Time for novel anticancer strategies?
Pharmacol Res. 2015; 100:288-95 [PubMed] Related Publications
Disruption of the circadian clock is associated with a variety of human pathologies, including cancer. Rather than being a mere consequence of a global changes associated with the cancer cell transcriptome, the aberrant clock gene expression observed in many tumors may serve for cancer cell survival. This scenario suggests the provocative hypothesis that pharmacological modulation of clock-related proteins may be suitable as an effective anticancer strategy. In this review, we focus on the functions of the druggable circadian nuclear receptors, REV-ERBα and REV-ERBβ, in cancer cell survival and describe the potential development of small molecule compounds that modulate REV-ERB activity as novel anticancer therapeutics. In addition, we debate the use of circadian rhythm-based synthetic lethal approaches to identify yet unexplored anticancer strategies.

Innominato PF, Lim AS, Palesh O, et al.
The effect of melatonin on sleep and quality of life in patients with advanced breast cancer.
Support Care Cancer. 2016; 24(3):1097-105 [PubMed] Related Publications
BACKGROUND: Fatigue and sleep problems are prevalent in cancer patients and can be associated with disruption of circadian rhythmicity. In this prospective phase II trial, we sought to assess the effect of melatonin on circadian biomarkers, sleep, and quality of life in breast cancer patients.
METHODS: Thirty-two patients with metastatic breast cancer, receiving hormonal or trastuzumab therapy, took 5 mg of melatonin at bedtime for 2 months. Before starting and after 2 months on melatonin therapy, sleep and circadian rhythmicity were assessed by actigraphy, diurnal patterns of serum cortisol, and the expression of the core clock genes PER2 and BMAL1 in peripheral blood mononuclear cells. The European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire was completed for subjective parameters.
RESULTS: Bedtime melatonin was associated with a significant improvement in a marker of objective sleep quality, sleep fragmentation and quantity, subjective sleep, fatigue severity, global quality of life, and social and cognitive functioning scales. Morning clock gene expression was increased following bedtime melatonin intake. Melatonin did not affect actigraphy measure of circadian rhythmicity, or the diurnal cortisol pattern.
CONCLUSION: These results invite further investigation of melatonin as a potentially useful therapeutic agent for improving sleep and quality of life in cancer patients.

Salavaty A
Carcinogenic effects of circadian disruption: an epigenetic viewpoint.
Chin J Cancer. 2015; 34(9):375-83 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
Circadian rhythms refer to the endogenous rhythms that are generated to synchronize physiology and behavior with 24-h environmental cues. These rhythms are regulated by both external cues and molecular clock mechanisms in almost all cells. Disruption of circadian rhythms, which is called circadian disruption, affects many biological processes within the body and results in different long-term diseases, including cancer. Circadian regulatory pathways result in rhythmic epigenetic modifications and the formation of circadian epigenomes. Aberrant epigenetic modifications, such as hypermethylation, due to circadian disruption may be involved in the transformation of normal cells into cancer cells. Several studies have indicated an epigenetic basis for the carcinogenic effects of circadian disruption. In this review, I first discuss some of the circadian genes and regulatory proteins. Then, I summarize the current evidence related to the epigenetic modifications that result in circadian disruption. In addition, I explain the carcinogenic effects of circadian disruption and highlight its potential role in different human cancers using an epigenetic viewpoint. Finally, the importance of chronotherapy in cancer treatment is highlighted.

Kochan DZ, Kovalchuk O
Circadian disruption and breast cancer: an epigenetic link?
Oncotarget. 2015; 6(19):16866-82 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
Breast cancer is already the most common malignancy affecting women worldwide, and evidence is mounting that breast cancer induced by circadian disruption (CD) is a warranted concern. Numerous studies have investigated various aspects of the circadian clock in relation to breast cancer, and evidence from these studies indicates that melatonin and the core clock genes can play a crucial role in breast cancer development. Even though epigenetics has been increasingly recognized as a key player in the etiology of breast cancer and linked to circadian rhythms, and there is evidence of overlap between epigenetic deregulation and breast cancer induced by circadian disruption, only a handful of studies have directly investigated the role of epigenetics in CD-induced breast cancer. This review explores the circadian clock and breast cancer, and the growing role of epigenetics in breast cancer development and circadian rhythms. We also summarize the current knowledge and next steps for the investigation of the epigenetic link in CD-induced breast cancer.

Dulong S, Ballesta A, Okyar A, Lévi F
Identification of Circadian Determinants of Cancer Chronotherapy through In Vitro Chronopharmacology and Mathematical Modeling.
Mol Cancer Ther. 2015; 14(9):2154-64 [PubMed] Related Publications
Cancer chronotherapy aims at enhancing tolerability and efficacy of anticancer drugs through their delivery according to circadian clocks. However, mouse and patient data show that lifestyle, sex, genetics, drugs, and cancer can modify both host circadian clocks and metabolism pathways dynamics, and thus the optimal timing of drug administration. The mathematical modeling of chronopharmacology could indeed help moderate optimal timing according to patient-specific determinants. Here, we combine in vitro and in silico methods, in order to characterize the critical molecular pathways that drive the chronopharmacology of irinotecan, a topoisomerase I inhibitor with complex metabolism and known activity against colorectal cancer. Large transcription rhythms moderated drug bioactivation, detoxification, transport, and target in synchronized colorectal cancer cell cultures. These molecular rhythms translated into statistically significant changes in pharmacokinetics and pharmacodynamics according to in vitro circadian drug timing. The top-up of the multiple coordinated chronopharmacology pathways resulted in a four-fold difference in irinotecan-induced apoptosis according to drug timing. Irinotecan cytotoxicity was directly linked to clock gene BMAL1 expression: The least apoptosis resulted from drug exposure near BMAL1 mRNA nadir (P < 0.001), whereas clock silencing through siBMAL1 exposure ablated all the chronopharmacology mechanisms. Mathematical modeling highlighted circadian bioactivation and detoxification as the most critical determinants of irinotecan chronopharmacology. In vitro-in silico systems chronopharmacology is a new powerful methodology for identifying the main mechanisms at work in order to optimize circadian drug delivery.

Lehmann R, Childs L, Thomas P, et al.
Assembly of a comprehensive regulatory network for the mammalian circadian clock: a bioinformatics approach.
PLoS One. 2015; 10(5):e0126283 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
By regulating the timing of cellular processes, the circadian clock provides a way to adapt physiology and behaviour to the geophysical time. In mammals, a light-entrainable master clock located in the suprachiasmatic nucleus (SCN) controls peripheral clocks that are present in virtually every body cell. Defective circadian timing is associated with several pathologies such as cancer and metabolic and sleep disorders. To better understand the circadian regulation of cellular processes, we developed a bioinformatics pipeline encompassing the analysis of high-throughput data sets and the exploitation of published knowledge by text-mining. We identified 118 novel potential clock-regulated genes and integrated them into an existing high-quality circadian network, generating the to-date most comprehensive network of circadian regulated genes (NCRG). To validate particular elements in our network, we assessed publicly available ChIP-seq data for BMAL1, REV-ERBα/β and RORα/γ proteins and found strong evidence for circadian regulation of Elavl1, Nme1, Dhx6, Med1 and Rbbp7 all of which are involved in the regulation of tumourigenesis. Furthermore, we identified Ncl and Ddx6, as targets of RORγ and REV-ERBα, β, respectively. Most interestingly, these genes were also reported to be involved in miRNA regulation; in particular, NCL regulates several miRNAs, all involved in cancer aggressiveness. Thus, NCL represents a novel potential link via which the circadian clock, and specifically RORγ, regulates the expression of miRNAs, with particular consequences in breast cancer progression. Our findings bring us one step forward towards a mechanistic understanding of mammalian circadian regulation, and provide further evidence of the influence of circadian deregulation in cancer.

Hill SM, Belancio VP, Dauchy RT, et al.
Melatonin: an inhibitor of breast cancer.
Endocr Relat Cancer. 2015; 22(3):R183-204 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
The present review discusses recent work on melatonin-mediated circadian regulation, the metabolic and molecular signaling mechanisms that are involved in human breast cancer growth, and the associated consequences of circadian disruption by exposure to light at night (LEN). The anti-cancer actions of the circadian melatonin signal in human breast cancer cell lines and xenografts heavily involve MT1 receptor-mediated mechanisms. In estrogen receptor alpha (ERα)-positive human breast cancer, melatonin suppresses ERα mRNA expression and ERα transcriptional activity via the MT1 receptor. Melatonin also regulates the transactivation of other members of the nuclear receptor superfamily, estrogen-metabolizing enzymes, and the expression of core clock and clock-related genes. Furthermore, melatonin also suppresses tumor aerobic metabolism (the Warburg effect) and, subsequently, cell-signaling pathways critical to cell proliferation, cell survival, metastasis, and drug resistance. Melatonin demonstrates both cytostatic and cytotoxic activity in breast cancer cells that appears to be cell type-specific. Melatonin also possesses anti-invasive/anti-metastatic actions that involve multiple pathways, including inhibition of p38 MAPK and repression of epithelial-mesenchymal transition (EMT). Studies have demonstrated that melatonin promotes genomic stability by inhibiting the expression of LINE-1 retrotransposons. Finally, research in animal and human models has indicated that LEN-induced disruption of the circadian nocturnal melatonin signal promotes the growth, metabolism, and signaling of human breast cancer and drives breast tumors to endocrine and chemotherapeutic resistance. These data provide the strongest understanding and support of the mechanisms that underpin the epidemiologic demonstration of elevated breast cancer risk in night-shift workers and other individuals who are increasingly exposed to LEN.

Chitikova Z, Pusztaszeri M, Makhlouf AM, et al.
Identification of new biomarkers for human papillary thyroid carcinoma employing NanoString analysis.
Oncotarget. 2015; 6(13):10978-93 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
We previously reported an upregulation of the clock transcript BMAL1, correlating with TIMP1 expression in fresh-frozen samples from papillary thyroid carcinoma (PTC). Since frozen postoperative biopsy samples are difficult to obtain, we aimed to validate the application of high-precision NanoString analysis for formalin-fixed paraffin-embedded (FFPE) thyroid nodule samples and to screen for potential biomarkers associated with PTC. No significant differences were detected between fresh-frozen and FFPE samples. NanoString analysis of 51 transcripts in 17 PTC and 17 benign nodule samples obtained from different donors and in 24 pairs of benign and PTC nodules, obtained from the same donor (multinodular goiters), confirmed significant alterations in the levels of BMAL1, c-MET, c-KIT, TIMP1, and other transcripts. Moreover, we identified for the first time alterations in CHEK1 and BCL2 levels in PTC. A predictive score was established for each sample, based on the combined expression levels of BMAL1, CHEK1, c-MET, c-KIT and TIMP1. In combination with BRAF mutation analysis, this predictive score closely correlated with the clinicopathological characteristics of the analyzed thyroid nodules. Our study identified new thyroid transcripts with altered levels in PTC using the NanoString approach. A predictive score correlation coefficient might contribute to improve the preoperative diagnosis of thyroid nodules.

Fang L, Yang Z, Zhou J, et al.
Circadian Clock Gene CRY2 Degradation Is Involved in Chemoresistance of Colorectal Cancer.
Mol Cancer Ther. 2015; 14(6):1476-87 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
Biomarkers for predicting chemotherapy response are important to the treatment of colorectal cancer patients. Cryptochrome 2 (CRY2) is a circadian clock protein involved in cell cycle, but the biologic consequences of this activity in cancer are poorly understood. We set up biochemical and cell biology analyses to analyze CRY2 expression and chemoresistance. Here, we report that CRY2 is overexpressed in chemoresistant colorectal cancer samples, and CRY2 overexpression is correlated with poor patient survival. Knockdown of CRY2 increased colorectal cancer sensitivity to oxaliplatin in colorectal cancer cells. We also identify FBXW7 as a novel E3 ubiquitin ligase for targeting CRY2 through proteasomal degradation. Mechanistic studies show that CRY2 is regulated by FBXW7, in which FBXW7 binds directly to phosphorylated Thr300 of CRY2. Furthermore, FBXW7 expression leads to degradation of CRY2 through enhancing CRY2 ubiquitination and accelerating the CRY2's turnover rate. High FBXW7 expression downregulates CRY2 and increases colorectal cancer cells' sensitivity to chemotherapy. Low FBXW7 expression is correlated with high CRY2 expression in colorectal cancer patient samples. Also, low FBXW7 expression is correlated with poor patient survival. Taken together, our findings indicate that the upregulation of CRY2 caused by downregulation of FBXW7 may be a novel prognostic biomarker and may represent a new therapeutic target in colorectal cancer.

Tavano F, Pazienza V, Fontana A, et al.
SIRT1 and circadian gene expression in pancreatic ductal adenocarcinoma: Effect of starvation.
Chronobiol Int. 2015; 32(4):497-512 [PubMed] Related Publications
Pancreatic cancer (PC), the fourth leading cause of cancer-related deaths, is characterized by high aggressiveness and resistance to chemotherapy. Pancreatic carcinogenesis is kept going by derangement of essential cell processes, such as proliferation, apoptosis, metabolism and autophagy, characterized by rhythmic variations with 24-h periodicity driven by the biological clock. We assessed the expression of the circadian genes ARNLT, ARNLT2, CLOCK, PER1, PER2, PER3, CRY1, CRY2 and the starvation-activated histone/protein deacetylase SIRT1 in 34 matched tumor and non-tumor tissue specimens of PC patients, and evaluated in PC derived cell lines if the modulation of SIRT1 expression through starvation could influence the temporal pattern of expression of the circadian genes. We found a significant down-regulation of ARNLT (p = 0.015), CRY1 (p = 0.013), CRY2 (p = 0.001), PER1 (p < 0.0001), PER2 (p < 0.001), PER3 (p = 0.001) and SIRT1 (p = 0.017) in PC specimens. PER3 and CRY2 expression levels were lower in patients with jaundice at diagnosis ( < 0.05). Having adjusted for age, adjuvant therapy and tumor stage, we evidenced that patients with higher PER2 and lower SIRT1 expression levels showed lower mortality (p = 0.028). Levels and temporal patterns of expression of many circadian genes and SIRT1 significantly changed upon serum starvation in vitro, with differences among four different PC cell lines examined (BXPC3, CFPAC, MIA-PaCa-2 and PANC-1). Serum deprivation induced changes of the overall mean level of the wave and amplitude, lengthened or shortened the cycle time and phase-advanced or phase-delayed the rhythmic oscillation depending on the gene and the PC cell line examined. In conclusion, a severe deregulation of expression of SIRT1 and circadian genes was evidenced in the cancer specimens of PC patients, and starvation influenced gene expression in PC cell lines, suggesting that the altered interplay between SIRT1 and the core circadian proteins could represent a crucial player in the process of pancreatic carcinogenesis.

Wang F, Luo Y, Li C, Chen L
Correlation between deregulated expression of PER2 gene and degree of glioma malignancy.
Tumori. 2014 Nov-Dec; 100(6):e266-72 [PubMed] Related Publications
AIMS AND BACKGROUND: Growing evidence indicates that disruption of circadian rhythms may be a risk factor for the development of glioma. However, the molecular mechanisms underlying the genetic regulation of circadian rhythms in glioma cells have yet to be explored.
METHODS AND STUDY DESIGN: Using immunohistochemical staining and methylation-specific PCR techniques, we examined the expression of the period 2 (PER2) gene, one of the most important clock genes, epidermal growth factor receptor (EGFR), and proliferating cell nuclear antigen (PCNA) in 92 gliomas.
RESULTS: Our results revealed disturbances in the expression of PER2 in most (52.17%) glioma cells compared with the expression in nearby noncancerous cells, and indicated that PER2 gene deregulation most likely occurs via methylation of PER2 promoters. The protein expression of PCNA and EGFR was significantly higher in high-grade than low-grade gliomas (P < 0.05). Furthermore, a negative correlation was detected between the protein expression of PER2 and PCNA and EGFR in glioma.
CONCLUSIONS: Because the circadian clock regulates the expression of cell cycle-related genes, we suggest that disturbances in PER2 gene expression may disrupt the regulation of the circadian clock, thus enhancing the survival of cancer cells and promoting carcinogenesis.

Ozden S, Tiber PM, Ozgen Z, et al.
Expression of TRF2 and its prognostic relevance in advanced stage cervical cancer patients.
Biol Res. 2014; 47:61 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
BACKGROUND: Telomeres are protective caps consisted of specific tandem repeats (5'-TTAGGG-3'). Shortening of telomeres at each cell division is known as "mitotic clock" of the cells, which renders telomeres as important regulators of lifespan. TRF2 is one of the critical members of shelterin complex, which is a protein complex responsible from the preservation of cap structure, and loss or mutation of TRF2 results in DNA damage, senescence or apoptosis. Since cancer is frequently associated with aberrant cell cycle progression, defective DNA repair or apoptosis pathways, TRF2 could be one likely candidate for cancer therapy. Here we investigated the prognostic role of TRF2 levels in cervical cancer patients. Fold-induction rates were evaluated with respect to median values after real-time PCR analysis. Overall survival, distant disease-free and local recurrence-free survival rates were calculated using Kaplan-Meier long rank test.
RESULTS: Both five year overall- and disease-free survival rates were longer in patients with higher TRF2 expression compared to lower expression, but results were not statistically significant (69.2% vs 28.9%, respectively). Mean local recurrence-free survivals (LRF) were very close ( 58.6, CI: 44.3-72.9 vs 54.5, CI: 32.1-76.9 months) for high and low expressions, respectively. Cumulative proportion of LRF at the end of five year period was 76.9% for high and 57.1% for low TRF2 expression (P = 0.75). Statistically significant difference was found between survival ratios and Bcl-xL and p53 gene expressions, but not with TRF2. A respectable correlation between TRF2 expression and apoptosis along with distant metastasis was noted (P = 0.045 and 0.036, respectively). Additionally, high TRF2 expression levels had a positive impact in five year survival rate of stage IIIB-IVA patients (P = 0.04).
CONCLUSIONS: Our results support the role of TRF2 in apoptosis and imply a positive relation with distant metastases and survival in advanced stage patients. The remarkable difference in survival periods of patients with different TRF2 expressions suggest that TRF2 may be a candidate factor to estimate survival for cervical cancer, a preliminary observation which should further be verified with a larger cohort.

Wang Y, Qian R, Sun N, et al.
Circadian gene hClock enhances proliferation and inhibits apoptosis of human colorectal carcinoma cells in vitro and in vivo.
Mol Med Rep. 2015; 11(6):4204-10 [PubMed] Article available free on PMC after 25/03/2017 Related Publications
Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy‑associated mortality worldwide. Previous studies have demonstrated that amplification and overexpression of the human circadian locomotor output cycles kaput gene (hClock) was closely associated with a high risk for CRC as well as poor prognosis in CRC patients. However, the underlying molecular mechanisms of CRC remain to be fully elucidated. In the present study, hClock was exogenously overexpressed in the CRC cell line SW480 via infection of a lentivirus vector expressing hClock; in addition, a lentivirus vector‑based RNA interference approach, using short hairpin RNA, was performed in order to knockdown hClock in SW620 cells. The results showed that upregulation of hClock promoted proliferation and inhibited apoptosis in SW480 cells in vitro and in vivo, while downregulation of hClock inhibited SW620 cell proliferation and accelerated apoptosis in vitro. Upregulation of hClock enhanced the activity of the anti‑apoptotic gene phosphorpylated (p‑)AKT and inhibited the expression of the pro‑apoptotic gene B cell lymphoma‑2 (Bcl‑2)‑associated X protein and Bcl‑2 homology 3 interacting domain death agonist. Furthermore, targeted inhibition of hClock activity reduced p‑AKT expression. In conclusion, the results of the present study suggested that the circadian gene hClock promoted CRC progression and inhibit tumor cell apoptosis in vitro and in vivo, while silencing hClock was able to reverse this effect.

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