Gene Summary

Gene:CLOCK; clock circadian regulator
Aliases: KAT13D, bHLHe8
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:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:circadian locomoter output cycles protein kaput
Source:NCBIAccessed: 01 September, 2019


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 (1994-2019)
Graph generated 01 September 2019 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.

  • Gene Expression Profiling
  • Aging
  • Cell Proliferation
  • Cell Cycle
  • Regression Analysis
  • Young Adult
  • Apoptosis
  • Transcription Factors
  • Neoplastic Cell Transformation
  • Genetic Predisposition
  • Biological Clocks
  • CLOCK Proteins
  • AKT1
  • Tandem Repeat Sequences
  • Nuclear Proteins
  • Circadian Clocks
  • Period Circadian Proteins
  • Epigenetics
  • Cryptochromes
  • Stomach Cancer
  • Proto-Oncogene Proteins c-myc
  • Virus Replication
  • Transcription
  • Circadian Rhythm
  • Messenger RNA
  • Chromosome 4
  • Cell Cycle Proteins
  • Cancer Gene Expression Regulation
  • Telomerase
  • Breast Cancer
  • Gene Expression Regulation
  • Biomarkers, Tumor
  • Colorectal Cancer
  • Ultraviolet Rays
  • Case-Control Studies
  • Sjogren's Syndrome
  • DNA Methylation
  • Suprachiasmatic Nucleus
  • ARNTL Transcription Factors
Tag cloud generated 01 September, 2019 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)

Tadepalli SH, Shields CL, Shields JA, Honavar SG
Intraocular medulloepithelioma - A review of clinical features, DICER 1 mutation, and management.
Indian J Ophthalmol. 2019; 67(6):755-762 [PubMed] Free Access to Full Article Related Publications
Intraocular medulloepithelioma is a nonhereditary neoplasm of childhood arising from primitive medullary epithelium. It most often involves the ciliary body. Most patients present between 2 and 10 years of age with loss of vision, pain, leucocoria, or conjunctival congestion. The mass appears as a grey-white ciliary body lesion with intratumoral cysts. Presence of a neoplastic cyclitic membrane with extension to retrolental region is characteristic. Secondary manifestations like cataract and neovascular glaucoma may be present in up to 50% and 60% patients, respectively. These could be the first signs for which, unfortunately, about 50% patients undergo surgery before recognition of the hidden tumor. Systemic correlation with pleuropulmonary blastoma (DICER1 gene) has been documented in 5% cases. Histopathology shows primitive neuroepithelial cells arranged as cords closely resembling the primitive retina. Histopathologically, the tumor is classified as teratoid (containing heteroplastic elements) and nonteratoid (containing medullary epithelial elements), each of which are further subclassified as benign or malignant. Retinoblastoma-like and sarcoma-like areas may be seen within the tissue. The treatment modality depends on tumor size and extent of invasion. For small localized tumors (≤3-4 clock hours), conservative treatments with cryotherapy, plaque radiotherapy, or partial lamellar sclerouvectomy (PLSU) have been used. Plaque brachytherapy is generally preferred for best tumor control. Advanced and extensive tumors require enucleation. Rare use of intra-arterial and intravitreal chemotherapy has been employed. Systemic prognosis is favorable, but those with extraocular extension and orbital involvement show risk for local recurrence and metastatic disease, which can lead to death.

Genov N, Basti A, Abreu M, Relógio A
Temporal Splicing Switches in Elements of the TNF-Pathway Identified by Computational Analysis of Transcriptome Data for Human Cell Lines.
Int J Mol Sci. 2019; 20(5) [PubMed] Free Access to Full Article Related Publications
Alternative splicing plays an important role in numerous cellular processes and aberrant splice decisions are associated with cancer. Although some studies point to a regulation of alternative splicing and its effector mechanisms in a time-dependent manner, the extent and consequences of such a regulation remains poorly understood. In the present work, we investigated the time-dependent production of isoforms in two Hodgkin lymphoma cell lines of different progression stages (HD-MY-Z, stage IIIb and L-1236, stage IV) compared to a B lymphoblastoid cell line (LCL-HO) with a focus on tumour necrosis factor (TNF) pathway-related elements. For this, we used newly generated time-course RNA-sequencing data from the mentioned cell lines and applied a computational pipeline to identify genes with isoform-switching behaviour in time. We analysed the temporal profiles of the identified events and evaluated in detail the potential functional implications of alterations in isoform expression for the selected top-switching genes. Our data indicate that elements within the TNF pathway undergo a time-dependent variation in isoform production with a putative impact on cell migration, proliferation and apoptosis. These include the genes

Rida P, Syed MI, Aneja R
Time will tell: Circadian clock dysregulation in triple negative breast cancer.
Front Biosci (Schol Ed). 2019; 11:178-192 [PubMed] Related Publications
Growing evidence now links circadian disruption (CD) to increased risk of developing multiple types of cancer, including breast cancer (BC). In the US, African-American (AA) BC patients have a higher mortality rate than European-Americans (EAs) with BC, and a prime suspect in this racially disparate burden has been the greater incidence of an aggressive and highly heterogeneous BC subtype called triple-negative BC (TNBC), among AAs. AAs are also more prone to CD as larger proportions of AAs engage in night shift work than EAs, and the chronotype of AAs makes it harder for them to adapt to CD than EAs. Although clock gene dysregulation has been shown to perturb transactivation of key cell cycle and apoptosis regulators, little is known about how clock gene mis-expression affects TNBC outcomes. This review examines the prognostic value of clock genes in TNBC, and evaluates patterns of clock gene dysregulation in the individual TNBC molecular subtypes. Better understanding of how CD contributes to TNBC biology may illuminate new paths to improving disease outcomes and reducing BC-related racial disparities.

Orhan T, Nielsen PB, Hviid TVF, et al.
Expression of Circadian Clock Genes in Human Colorectal Cancer Tissues Using Droplet Digital PCR.
Cancer Invest. 2019; 37(2):90-98 [PubMed] Related Publications
Increasing evidence indicates that disruption of circadian rhythms may be directly linked to cancer. Here we report that the expression levels of the core clock genes Per1 and Per3 measured by droplet digital polymerase chain reaction are significantly decreased in tumour tissue from 16 patients undergoing colorectal cancer surgery compared to paired normal mucosa. No differences were observed in the expression of Per2, Bmal1, and Clock. In conclusion, abnormal expression levels of the clock genes Per1 and Per3 in CRC tissue may be related to tumourigenesis and may provide future diagnostic and prognostic information.

Peng H, Zhang J, Zhang PP, et al.
ARNTL hypermethylation promotes tumorigenesis and inhibits cisplatin sensitivity by activating CDK5 transcription in nasopharyngeal carcinoma.
J Exp Clin Cancer Res. 2019; 38(1):11 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Increasing evidence support an important role for DNA methylation in nasopharyngeal carcinoma (NPC). Here, we explored the role of circadian clock gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL) methylation in NPC.
METHODS: We employed bisulfite pyrosequencing to determine the epigenetic change of ARNTL in NPC cell lines and tissues. ARNTL mRNA and protein expression in cell lines and tissues were detected by real-time PCR and western blotting. Then, we constructed cell lines overexpressing ARNTL and knocked down ARNTL to explore its function and effect on chemotherapy sensitivity of NPC cell lines to cisplatin in vitro and vivo. Finally, we investigated the potential molecular mechanism of ARNTL by gene set enrichment analysis (GSEA), dual Luciferase reporter assay and chromatin immunoprecipitation assay.
RESULTS: ARNTL was hypermethylated, and its mRNA and protein were significantly down-regulated in NPC cell lines and tissues. When treated by 5-aza-2'-deoxycytidine, mRNA expression was up-regulated. Overexpression of ARNTL could suppress NPC cells proliferation in vitro and vivo while silencing of ARNTL using shRNA achieved opposite results. GSEA assay found that ARNTL was associated with cell cycle and ectopic ARNTL overexpression could induce G2-M phase arrest. Then, we identified and validated cyclin-dependent kinase 5 (CDK5) as the targeting gene of ARNTL by dual Luciferase reporter assay and chromatin immunoprecipitation assay. When transiently infected ARNTL-overexpression cells with PENTER-vector or PENTER-CDK5 plasmids, the later could reverse the suppressive effects of ARNTL on NPC cell proliferation. Moreover, ARNTL significantly enhanced sensitivity to cisplatin in NPC cells.
CONCLUSIONS: ARNTL suppresses NPC cell proliferation and enhances sensitivity to cisplatin by targeting CDK5. ARNTL may represent a novel therapeutic target for NPC.

Matsumura R, Akashi M
Role of the clock gene Period3 in the human cell-autonomous circadian clock.
Genes Cells. 2019; 24(2):162-171 [PubMed] Related Publications
Previous studies have shown that mouse Period3 (mPer3) is dispensable for the generation of autonomous oscillations in the circadian clock. However, human studies have suggested that human Period3 (hPer3) may have more important roles in the core clock machinery than mPer3. To investigate the role of hPer3 protein in the cell-autonomous circadian oscillator, we conducted gene knockout of the hPer3 gene in human bone osteosarcoma epithelial cells using genome-editing technology. We examined the circadian transcription of endogenous clock genes in hPer3-deficient cell clones and found that hPer3-deficient cells showed a phase advance in circadian transcription compared to wild-type cells. We subsequently transfected wild-type and mutant cells with an adenovirus carrying a luciferase gene whose expression was driven by a clock gene promotor, and monitored bioluminescence in real time. Cosinor analysis showed that the circadian period length in all hPer3-deficient cells was significantly shorter than that in wild-type cells, demonstrating that the phase advance in endogenous clock gene expression in hPer3-deficient cell clones was attributable to a shortened circadian period length rather than a phase shift. Together these findings are consistent with previous studies in mice lacking functional mPer3, indicating that the Per3 protein functions similarly in both mice and humans.

Masri S, Sassone-Corsi P
The emerging link between cancer, metabolism, and circadian rhythms.
Nat Med. 2018; 24(12):1795-1803 [PubMed] Free Access to Full Article Related Publications
The circadian clock is a complex cellular mechanism that, through the control of diverse metabolic and gene expression pathways, governs a large array of cyclic physiological processes. Epidemiological and clinical data reveal a connection between the disruption of circadian rhythms and cancer that is supported by recent preclinical data. In addition, results from animal models and molecular studies underscore emerging links between cancer metabolism and the circadian clock. This has implications for therapeutic approaches, and we discuss the possible design of chronopharmacological strategies.

Gerhauser C, Favero F, Risch T, et al.
Molecular Evolution of Early-Onset Prostate Cancer Identifies Molecular Risk Markers and Clinical Trajectories.
Cancer Cell. 2018; 34(6):996-1011.e8 [PubMed] Related Publications
Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ≤55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.

Benna C, Rajendran S, Spiro G, et al.
Associations of clock genes polymorphisms with soft tissue sarcoma susceptibility and prognosis.
J Transl Med. 2018; 16(1):338 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Dysfunction of the circadian clock and polymorphisms of some circadian genes have been linked to cancer development and progression. We investigated the relationship between circadian genes germline variation and susceptibility or prognosis of patients with soft tissue sarcoma.
PATIENTS AND METHODS: We considered the 14 single nucleotide polymorphisms (SNPs) of 6 core circadian genes that have a minor allele frequency > 5% and that are known to be associated with cancer risk or prognosis. Genotyping was performed by q-PCR. Peripheral blood and clinic-pathological data were available for 162 patients with liposarcoma or leiomyosarcoma and 610 healthy donors. Associations between the selected clock genes polymorphisms and sarcoma susceptibility or prognosis were tested assuming 3 models of inheritance: additive, recessive and dominant. Subgroup analysis based on sarcoma histotype was performed under the additive genetic model. Multivariate logistic regression and multivariate Cox proportional hazard regression analyses were utilized to assess the association between SNPs with patient susceptibility and survival, respectively. Pathway variation analysis was conducted employing the Adaptive Rank Truncated Product method.
RESULTS: Six out of the 14 analyzed SNPs were statistically significantly associated with susceptibility or prognosis of soft tissue sarcoma (P < 0.05). The present analysis suggested that carriers of the minor allele of the CLOCK polymorphism rs1801260 (C) or of PER2 rs934945 (T) had a reduced predisposition to sarcoma (26% and 35% respectively with the additive model) and liposarcoma (33% and 41% respectively). The minor allele (A) of NPAS2 rs895520 was associated with an increased predisposition to sarcoma of 33% and leiomyosarcoma of 44%. RORA rs339972 C allele was associated with a decreased predisposition to develop sarcoma assuming an additive model (29%) and leiomyosarcoma (36%). PER1 rs3027178 was associated with a reduced predisposition only in liposarcoma subgroup (32%). rs7602358 located upstream PER2 was significantly associated with liposarcoma survival (HR: 1.98; 95% CI 1.02-3.85; P = 0.04). Germline genetic variation in the circadian pathway was associated with the risk of developing soft tissue sarcoma (P = 0.035).
CONCLUSIONS: Genetic variation of circadian genes appears to play a role in the determinism of patient susceptibility and prognosis. These findings prompt further studies to fully dissect the molecular mechanisms.

Broadberry E, McConnell J, Williams J, et al.
Disrupted circadian clocks and altered tissue mechanics in primary human breast tumours.
Breast Cancer Res. 2018; 20(1):125 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer.
METHODS: We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy.
RESULTS: Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia.
CONCLUSION: Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.

Kazemi Noureini S, Fatemi L, Wink M
Telomere shortening in breast cancer cells (MCF7) under treatment with low doses of the benzylisoquinoline alkaloid chelidonine.
PLoS One. 2018; 13(10):e0204901 [PubMed] Free Access to Full Article Related Publications
Telomeres, the specialized dynamic structures at chromosome ends, regularly shrink with every replication. Thus, they function as an internal molecular clock counting down the number of cell divisions. However, most cancer cells escape this limitation by activating telomerase, which can maintain telomere length. Previous studies showed that the benzylisoquinoline alkaloid chelidonine stimulates multiple modes of cell death and strongly down-regulates telomerase. It is still unknown if down-regulation of telomerase by chelidonine boosts substantial telomere shortening. The breast cancer cell line MCF7 was sequentially treated with very low concentrations of chelidonine over several cell passages. Telomere length and telomerase activity were measured by a monochrome multiplex quantitative PCR and a q-TRAP assay, respectively. Changes in population size and doubling time correlated well with telomerase inhibition and telomere shortening. MCF7 cell growth was arrested completely after three sequential treatments with 0.1 μM chelidonine, each ending after 48 h, while telomere length was reduced to almost 10% of the untreated control. However, treatment with 0.01 μM chelidonine did not have any apparent consequence. In addition to dose and time dependent telomerase inhibition, chelidonine changed the splicing pattern of hTERT towards non-enzyme coding isoforms of the transcript. In conclusion, telomere length and telomere stability are strongly affected by chelidonine in addition to microtubule formation.

Saldivar JC, Park D
Mechanisms shaping the mutational landscape of the FRA3B/FHIT-deficient cancer genome.
Genes Chromosomes Cancer. 2019; 58(5):317-323 [PubMed] Related Publications
Genome instability is an enabling characteristic of cancer that facilitates the acquisition of oncogenic mutations that drive tumorigenesis. Underlying much of the instability in cancer is DNA replication stress, which causes both chromosome structural changes and single base-pair mutations. Common fragile sites are some of the earliest and most frequently altered loci in tumors. Notably, the fragile locus, FRA3B, lies within the fragile histidine triad (FHIT) gene, and consequently deletions within FHIT are common in cancer. We review the evidence in support of FHIT as a DNA caretaker and discuss the mechanism by which FHIT promotes genome stability. FHIT increases thymidine kinase 1 (TK1) translation to balance the deoxyribonucleotide triphosphates (dNTPs) for efficient DNA replication. Consequently, FHIT-loss causes replication stress, DNA breaks, aneuploidy, copy-number changes (CNCs), small insertions and deletions, and point mutations. Moreover, FHIT-loss-induced replication stress and DNA breaks cooperate with APOBEC3B overexpression to catalyze DNA hypermutation in cancer, as APOBEC family enzymes prefer single-stranded DNA (ssDNA) as substrates and ssDNA is enriched at sites of both replication stress and DNA breaks. Consistent with the frequent loss of FHIT across a broad spectrum of cancer types, FHIT-deficiency is highly associated with the ubiquitous, clock-like mutation signature 5 occurring in all cancer types thus far examined. The ongoing destabilization of the genome caused by FHIT loss underlies recurrent inactivation of tumor suppressors and activation of oncogenes. Considering that more than 50% of cancers are FHIT-deficient, we propose that FRA3B/FHIT fragility shapes the mutational landscape of cancer genomes.

Phillips DH
Mutational spectra and mutational signatures: Insights into cancer aetiology and mechanisms of DNA damage and repair.
DNA Repair (Amst). 2018; 71:6-11 [PubMed] Free Access to Full Article Related Publications
Reporter gene assays, in which a single mutation from each experiment can contribute to the assembly of a mutation spectrum for an agent, have provided the basis for understanding the mutational processes induced by mutagenic agents and for providing clues to the origins of mutations in human tumours. More recently exome and whole genome sequencing of human tumours has revealed distinct patterns of mutation that could provide additional clues for the causative origins of cancer. This can be tested by examining the mutational signatures induced in experimental systems by putative cancer-causing agents. Such signatures are now being generated in vitro in a number of different mutagen-exposed cellular systems. Results reveal that mutagens induce characteristic mutation signatures that, in some cases, match signatures found in human tumours. Proof of principle has been established with mutational signatures generated by simulated sunlight and aristolochic acid, which match those signatures found in human melanomas and urothelial cancers, respectively. In an analysis of somatic mutations in cancers for which tobacco smoking confers an elevated risk, it was found that smoking is associated with increased mutation burdens of multiple different mutational signatures, which contribute to different extents in different tissues. One of these signatures, mainly found in tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA editing by APOBEC cytidine deaminases and of an endogenous clock-like mutational process. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load although direct evidence for this mechanism is lacking in some cancer types. Thus, next generation sequencing of exomes or whole genomes is providing new insights into processes underlying the causes of human cancer.

Xiang R, Cui Y, Wang Y, et al.
Circadian clock gene Per2 downregulation in non‑small cell lung cancer is associated with tumour progression and metastasis.
Oncol Rep. 2018; 40(5):3040-3048 [PubMed] Related Publications
Period2 (Per2) is a key circadian clock gene, and its deregulation contributes to tumour development, including breast cancer. However, the biological function and clinicopathological significance of Per2 in non‑small cell lung cancer (NSCLC) remain unclear. The present study aimed to explore the role of Per2 and its relative clinical significance in NSCLC. To analyse Per2 expression in NSCLC specimens, reverse transcription‑quantitative polymerase chain reaction was performed, and the results indicated that Per2 expression was markedly downregulated in 83.87% (26/31) of NSCLC samples compared with their adjacent matched tissues. Increased Per2 expression was associated with increased differentiation (P<0.01) and reduced lymph node metastasis (P<0.0001). Functional studies identified that enhancing Per2 expression in A549 cells by lentivirus transduction not only significantly suppressed cell growth, migration and invasion (P<0.05) but also inhibited NSCLC growth and metastasis in vivo. Animal studies and histopathological analysis identified that Per2 expression in A549 cells not only markedly increased expression of tumour anti‑oncogenes Bax, P53 and P21 but also inhibited expression of pro‑oncogenes vascular endothelial growth factor, CD44 and c‑Myc. These results indicate that the loss of Per2 is one of the factors underlying tumourigenesis in NSCLC, and it may function as a novel molecular target for NSCLC.

Reszka E, Zienolddiny S
Epigenetic Basis of Circadian Rhythm Disruption in Cancer.
Methods Mol Biol. 2018; 1856:173-201 [PubMed] Related Publications
Self-sustained and synchronized to environmental stimuli, circadian clocks are under genetic and epigenetic regulation. Recent findings have greatly increased our understanding of epigenetic plasticity governed by circadian clock. Thus, the link between circadian clock and epigenetic machinery is reciprocal. Circadian clock can affect epigenetic features including genomic DNA methylation, noncoding RNA, mainly miRNA expression, and histone modifications resulted in their 24-h rhythms. Concomitantly, these epigenetic events can directly modulate cyclic system of transcription and translation of core circadian genes and indirectly clock output genes. Significant findings interlocking circadian clock, epigenetics, and cancer have been revealed, particularly in breast, colorectal, and blood cancers. Aberrant methylation of circadian gene promoter regions and miRNA expression affected circadian gene expression, together with 24-h expression oscillation pace have been frequently observed.

Fuhr L, El-Athman R, Scrima R, et al.
The Circadian Clock Regulates Metabolic Phenotype Rewiring Via HKDC1 and Modulates Tumor Progression and Drug Response in Colorectal Cancer.
EBioMedicine. 2018; 33:105-121 [PubMed] Free Access to Full Article Related Publications
An endogenous molecular clockwork drives various cellular pathways including metabolism and the cell cycle. Its dysregulation is able to prompt pathological phenotypes including cancer. Besides dramatic metabolic alterations, cancer cells display severe changes in the clock phenotype with likely consequences in tumor progression and treatment response. In this study, we use a comprehensive systems-driven approach to investigate the effect of clock disruption on metabolic pathways and its impact on drug response in a cellular model of colon cancer progression. We identified distinctive time-related transcriptomic and metabolic features of a primary tumor and its metastatic counterpart. A mapping of the expression data to a comprehensive genome-scale reconstruction of human metabolism allowed for the in-depth functional characterization of 24 h-oscillating transcripts and pointed to a clock-driven metabolic reprogramming in tumorigenesis. In particular, we identified a set of five clock-regulated glycolysis genes, ALDH3A2, ALDOC, HKDC1, PCK2, and PDHB with differential temporal expression patterns. These findings were validated in organoids and in primary fibroblasts isolated from normal colon and colon adenocarcinoma from the same patient. We further identified a reciprocal connection of HKDC1 to the clock in the primary tumor, which is lost in the metastatic cells. Interestingly, a disruption of the core-clock gene BMAL1 impacts on HKDC1 and leads to a time-dependent rewiring of metabolism, namely an increase in glycolytic activity, as well as changes in treatment response. This work provides novel evidence regarding the complex interplay between the circadian clock and metabolic alterations in carcinogenesis and identifies new connections between both systems with pivotal roles in cancer progression and response to therapy.

Fages F, Martinez T, Rosenblueth DA, Soliman S
Influence Networks Compared with Reaction Networks: Semantics, Expressivity and Attractors.
IEEE/ACM Trans Comput Biol Bioinform. 2018 Jul-Aug; 15(4):1138-1151 [PubMed] Related Publications
Biochemical reaction networks are one of the most widely used formalisms in systems biology to describe the molecular mechanisms of high-level cell processes. However, modellers also reason with influence diagrams to represent the positive and negative influences between molecular species and may find an influence network useful in the process of building a reaction network. In this paper, we introduce a formalism of influence networks with forces, and equip it with a hierarchy of Boolean, Petri net, stochastic and differential semantics, similarly to reaction networks with rates. We show that the expressive power of influence networks is the same as that of reaction networks under the differential semantics, but weaker under the discrete semantics. Furthermore, the hierarchy of semantics leads us to consider a (positive) Boolean semantics that cannot test the absence of a species, that we compare with the (negative) Boolean semantics with test for absence of a species in gene regulatory networks à la Thomas. We study the monotonicity properties of the positive semantics and derive from them an algorithm to compute attractors in both the positive and negative Boolean semantics. We illustrate our results on models of the literature about the p53/Mdm2 DNA damage repair system, the circadian clock, and the influence of MAPK signaling on cell-fate decision in urinary bladder cancer.

Marwitz S, Heinbockel L, Scheufele S, et al.
Fountain of youth for squamous cell carcinomas? On the epigenetic age of non-small cell lung cancer and corresponding tumor-free lung tissues.
Int J Cancer. 2018; 143(12):3061-3070 [PubMed] Free Access to Full Article Related Publications
Aging affects the core processes of almost every organism, and the functional decline at the cellular and tissue levels influences disease development. Recently, it was shown that the methylation of certain CpG dinucleotides correlates with chronological age and that this epigenetic clock can be applied to study aging-related effects. We investigated these molecular age loci in non-small cell lung cancer (NSCLC) tissues from patients with adenocarcinomas (AC) and squamous cell carcinomas (SQC) as well as in matched tumor-free lung tissue. In both NSCLC subtypes, the calculated epigenetic age did not correlate with the chronological age. In particular, SQC exhibited rejuvenation compared to the corresponding normal lung tissue as well as with the chronological age of the donor. Moreover, the younger epigenetic pattern was associated with a trend toward stem cell-like gene expression patterns. These findings show deep phenotypic differences between the tumor entities AC and SQC, which might be useful for novel therapeutic and diagnostic approaches.

Lesicka M, Jabłońska E, Wieczorek E, et al.
Altered circadian genes expression in breast cancer tissue according to the clinical characteristics.
PLoS One. 2018; 13(6):e0199622 [PubMed] Free Access to Full Article Related Publications
Breast cancer has a multifactorial etiology. One of the supposed and novel mechanisms is an alteration of circadian gene expression. Circadian genes play a crucial role in many physiological processes. These processes, such as genomic stability, DNA repair mechanism and apoptosis, are frequently disrupted in breast tumors. To assess the significance of circadian gene expression in breast cancer, we carried out an analysis of CLOCK, BMAL1, NPAS2, PER1, PER2, PER3 and CRY1, CRY2, TIMELESS, CSNK1E expression by the use of the quantitative Real-Time PCR technique in tumor tissue and non-tumor adjacent normal tissue sampled from 107 women with a newly diagnosed disease. The obtained data were compared to the clinical and histopathological features. PER1, PER2, PER3, CRY2 were found to be significantly down-expressed, while CLOCK, TIMELESS were over-expressed in the studied tumor samples compared to the non-tumor samples. Only gene expression of CRY1 was significantly down-regulated with progression according to the TNM classification. We found significantly decreased expression of CRY2, PER1, PER2 genes in the ER/PR negative breast tumors compared to the ER/PR positive tumors. Additionally, expression of CRY2, NPAS2 genes had a decreased level in the poorly differentiated tumors in comparison with the well and moderately differentiated ones. Our results indicate that circadian gene expression is altered in breast cancer tissue, which confirms previous observations from various animal and in vitro studies.

Hasakova K, Vician M, Reis R, et al.
The expression of clock genes cry1 and cry2 in human colorectal cancer and tumor adjacent tissues correlates differently dependent on tumor location.
Neoplasma. 2018; 65(6):986-992 [PubMed] Related Publications
Colorectal cancer (CRC) exhibits differences in its features depending on the location of the tumor. The role of the circadian system in carcinogenesis is accepted, and many studies report different clock gene expression in tumors compared to healthy tissue. However, little attention is given to the changes in clock genes in tumors arising from various locations across the colon and rectum. The aim of our study was to investigate the expression of the clock genes cry1 and cry2 in human CRC tissue and tissue adjacent to colorectal tumors in a cohort of 64 patients by real time PCR. Expression of cry1 in the entire patient cohort was higher in tumors compared to adjacent tissues in the right-sided colon but not in the left-sided colorectum. Difference in cry1 expression between tumor and adjacent tissue in the right-sided colon was preserved in women and a trend was observed in men. Higher expression of cry1 in the right-sided colon tumor tissue was associated with worse survival in women and the expression of cry1 in the left-sided colorectum was significantly higher in the adjacent tissue compared to tumor in men but not in women. Expression of cry2 was lower in the tumor than in adjacent tissue in both the right and left-sided colorectum. This trend was generally preserved, but the difference reached significance level only in the male left-sided colon, and cry2 expression in the tumor tissue significantly correlated with location of the tumor in men with grade 2 cancer. Finally, we detected significant correlation between tumor location and cry1 expression in the adjacent tissue and the combined results establish that tumor influence on adjacent tissue is dependent on tumor location. Changed clock gene expression should therefore be considered in specific CRC patient sub-groups.

El-Athman R, Fuhr L, Relógio A
A Systems-Level Analysis Reveals Circadian Regulation of Splicing in Colorectal Cancer.
EBioMedicine. 2018; 33:68-81 [PubMed] Free Access to Full Article Related Publications
Accumulating evidence points to a significant role of the circadian clock in the regulation of splicing in various organisms, including mammals. Both dysregulated circadian rhythms and aberrant pre-mRNA splicing are frequently implicated in human disease, in particular in cancer. To investigate the role of the circadian clock in the regulation of splicing in a cancer progression context at the systems-level, we conducted a genome-wide analysis and compared the rhythmic transcriptional profiles of colon carcinoma cell lines SW480 and SW620, derived from primary and metastatic sites of the same patient, respectively. We identified spliceosome components and splicing factors with cell-specific circadian expression patterns including SRSF1, HNRNPLL, ESRP1, and RBM 8A, as well as altered alternative splicing events and circadian alternative splicing patterns of output genes (e.g., VEGFA, NCAM1, FGFR2, CD44) in our cellular model. Our data reveals a remarkable interplay between the circadian clock and pre-mRNA splicing with putative consequences in tumor progression and metastasis.

Wagner PM, Sosa Alderete LG, Gorné LD, et al.
Proliferative Glioblastoma Cancer Cells Exhibit Persisting Temporal Control of Metabolism and Display Differential Temporal Drug Susceptibility in Chemotherapy.
Mol Neurobiol. 2019; 56(2):1276-1292 [PubMed] Related Publications
Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes,

Yu Y, Li Y, Zhou L, et al.
Cryptochrome 2 (CRY2) Suppresses Proliferation and Migration and Regulates Clock Gene Network in Osteosarcoma Cells.
Med Sci Monit. 2018; 24:3856-3862 [PubMed] Free Access to Full Article Related Publications
BACKGROUND Circadian disruption is a potential cancer risk factor in humans. However, the role of the clock gene, cryptochrome 2 (CRY2), in osteosarcoma (OS) is still not clear. MATERIAL AND METHODS To evaluate the potential role of CRY2 in HOS osteosarcoma cells, CRY2-silenced cell lines were established. Furthermore, we investigated the effect of CRY2 knockdown on HOS cells by CCK-8, colony formation, migration assay, and flow cytometry, in vitro. RESULTS CRY2 knockdown promoted HOS OS cell proliferation and migration. We used a cell cycle assay to show that CRY2 knockdown increased the S phase cell population and reduced the G1 phase cell population. Western blot analyses showed that CRY2 knockdown decreased P53 expression and increased expression of c-myc and cyclin D1. Simultaneously, CRY2 knockdown increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, but did not change the phosphorylation of c-Jun N terminal kinase (JNK) and P38. CRY2 knockdown also increased the expression of matrix metalloproteinase (MMP)-2 and β-catenin, and increased OS cell proliferation and migration by inducing cell cycle progression and promoting mitogen-activated protein kinase (MAPK) and Wnt/β-catenin signaling pathways. Although it has previously been unclear whether the expression of CRY2 affects the expression of other clock genes in the clock gene network, our results show that knockdown of CRY2 significantly increased the mRNA expression of CRY1, Period (PER) 1, PER2, BMAL1, and CLOCK. CONCLUSIONS Our results suggest that CRY2 may be an anti-oncogene in OS, whose functions involve both downstream genes and other circadian genes.

Tetti M, Castellano I, Venziano F, et al.
Role of Cryptochrome-1 and Cryptochrome-2 in Aldosterone-Producing Adenomas and Adrenocortical Cells.
Int J Mol Sci. 2018; 19(6) [PubMed] Free Access to Full Article Related Publications
Mice lacking the core-clock components, cryptochrome-1 (CRY1) and cryptochrome-2 (CRY2) display a phenotype of hyperaldosteronism, due to the upregulation of type VI 3β-hydroxyl-steroid dehydrogenase (

Giudice A, Crispo A, Grimaldi M, et al.
The Effect of Light Exposure at Night (LAN) on Carcinogenesis via Decreased Nocturnal Melatonin Synthesis.
Molecules. 2018; 23(6) [PubMed] Free Access to Full Article Related Publications
In mammals, a master clock is located within the suprachiasmatic nucleus (SCN) of the hypothalamus, a region that receives input from the retina that is transmitted by the retinohypothalamic tract. The SCN controls the nocturnal synthesis of melatonin by the pineal gland that can influence the activity of the clock's genes and be involved in the inhibition of cancer development. On the other hand, in the literature, some papers highlight that artificial light exposure at night (LAN)-induced circadian disruptions promote cancer. In the present review, we summarize the potential mechanisms by which LAN-evoked disruption of the nocturnal increase in melatonin synthesis counteracts its preventive action on human cancer development and progression. In detail, we discuss: (i) the Warburg effect related to tumor metabolism modification; (ii) genomic instability associated with L1 activity; and (iii) regulation of immunity, including regulatory T cell (Treg) regulation and activity. A better understanding of these processes could significantly contribute to new treatment and prevention strategies against hormone-related cancer types.

Bellastella G, Scappaticcio L, Esposito K, et al.
Metabolic syndrome and cancer: "The common soil hypothesis".
Diabetes Res Clin Pract. 2018; 143:389-397 [PubMed] Related Publications
Metabolic syndrome (MetS) and cancer share many modifiable risk factors including age, genetic factors, obesity, physical inactivity, unhealthy diet, alcohol, smoking, endocrine disruptors exposure, circadian clock disturbances, and air pollution. MetS is closely linked to cancer, as it increases cancer risk and cancer-related mortality; moreover, cancer survivors have an increased risk of MetS. Elucidating the mechanisms linking MetS to cancer is important to prevent or delay these two conditions. Possible mechanisms explaining the relationship between MetS and cancer include hyperinsulinemia and alterations of insulin-like growth factor system, chronic subclinical inflammation, abnormalities in sex hormones metabolism and adipokines, hyperglicemia, alterations in both gene expression and hormonal profile by endocrine disruptors and air pollution, desynchronization of circadian clock. The common soil hypothesis claims that MetS may be considered a surrogate marker for dietary risk factors of cancer, and a warning sign for susceptible individuals exposed to an unhealthy diet. The common soil hypothesis and the clepsydra of foods represent a theoretical substrate to preventive intervention strategies against the pandemics of MetS and cancer: adherence to healthy dietary patterns is associated with a reduced risk of MetS, and improvement of the quality of diet is consistently associated with a reduction in cancer-related mortality.

Sánchez DI, González-Fernández B, Crespo I, et al.
Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine-induced hepatocellular carcinoma.
J Pineal Res. 2018; 65(3):e12506 [PubMed] Related Publications
Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long-term diseases including hepatocellular carcinoma (HCC). Melatonin has been reported to alleviate promotion and progression of HCC, but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg

Waszak SM, Northcott PA, Buchhalter I, et al.
Spectrum and prevalence of genetic predisposition in medulloblastoma: a retrospective genetic study and prospective validation in a clinical trial cohort.
Lancet Oncol. 2018; 19(6):785-798 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines.
METHODS: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MB
FINDINGS: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MB
INTERPRETATION: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MB
FUNDING: German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario.

Yang Y, Adebali O, Wu G, et al.
Cisplatin-DNA adduct repair of transcribed genes is controlled by two circadian programs in mouse tissues.
Proc Natl Acad Sci U S A. 2018; 115(21):E4777-E4785 [PubMed] Related Publications
Cisplatin is a major cancer chemotherapeutic drug. It kills cancer cells by damaging their DNA, mainly in the form of Pt-d(GpG) diadducts. However, it also has serious side effects, including nephrotoxicity and hepatotoxicity that limit its usefulness. Chronotherapy is taking circadian time into account during therapy to improve the therapeutic index, by improving efficacy and/or limiting toxicity. To this end, we tested the impact of clock time on excision repair of cisplatin-induced DNA damage at single-nucleotide resolution across the genome in mouse kidney and liver. We found that genome repair is controlled by two circadian programs. Repair of the transcribed strand (TS) of active, circadian-controlled genes is dictated by each gene's phase of transcription, which falls across the circadian cycle with prominent peaks at dawn and dusk. In contrast, repair of the nontranscribed strand (NTS) of all genes, repair of intergenic DNA, and global repair overall peaks at Zeitgeber time ZT08, as basal repair capacity, which is controlled by the circadian clock, peaks at this circadian time. Consequently, the TS and NTS of many genes are repaired out of phase. As most cancers are thought to have defective circadian rhythms, these results suggest that future research on timed dosage of cisplatin could potentially reduce damage to healthy tissue and improve its therapeutic index.

Matsunaga N, Ogino T, Hara Y, et al.
Optimized Dosing Schedule Based on Circadian Dynamics of Mouse Breast Cancer Stem Cells Improves the Antitumor Effects of Aldehyde Dehydrogenase Inhibitor.
Cancer Res. 2018; 78(13):3698-3708 [PubMed] Related Publications
Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and is used for identification and isolation of CSC. Here, we show that the population of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDH-positive cells were generated by time-dependent increases and decreases in the expression of

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