Disturbance of the Circadian System in Shift Work and Its Health Impact

The various non-standard schedules required of shift workers force abrupt changes in the timing of sleep and light-dark exposure. These changes result in disturbances of the endogenous circadian system and its misalignment with the environment. Simulated night-shift experiments and field-based studies with shift workers both indicate that the circadian system is resistant to adaptation from a day- to a night-oriented schedule, as determined by a lack of substantial phase shifts over multiple days in centrally controlled rhythms, such as those of melatonin and cortisol. There is evidence that disruption of the circadian system caused by night-shift work results not only in a misalignment between the circadian system and the external light-dark cycle, but also in a state of internal desynchronization between various levels of the circadian system. This is the case between rhythms controlled by the central circadian pacemaker and clock genes expression in tissues such as peripheral blood mononuclear cells, hair follicle cells, and oral mucosa cells. The disruptive effects of atypical work schedules extend beyond the expression profile of canonical circadian clock genes and affects other transcripts of the human genome. In general, after several days of living at night, most rhythmic transcripts in the human genome remain adjusted to a day-oriented schedule, with dampened group amplitudes. In contrast to circadian clock genes and rhythmic transcripts, metabolomics studies revealed that most metabolites shift by several hours when working nights, thus leading to their misalignment with the circadian system. Altogether, these circadian and sleep-wake disturbances emphasize the all-encompassing impact of night-shift work, and can contribute to the increased risk of various medical conditions. Here, we review the latest scientific evidence regarding the effects of atypical work schedules on the circadian system, sleep and alertness of shift-working populations, and discuss their potential clinical impacts.

Circadian clock regulates granulosa cell autophagy through NR1D1-mediated inhibition of ATG5

Autophagy of granulosa cells (GCs) is involved in follicular atresia, which occurs repeatedly during the ovarian development cycle. Several circadian clock genes are rhythmically expressed in both rodent ovarian tissues and GCs. Nuclear receptor subfamily 1 group D member 1 (NR1D1), an important component of the circadian clock system, is involved in the autophagy process through the regulation of autophagy-related genes. However, there are no reports illustrating the role of the circadian clock system in mouse GC autophagy. In the present study, we found that core circadian clock genes (Bmal1, Per2, Nr1d1, and Dbp) and an autophagy-related gene (Atg5) exhibited rhythmic expression patterns across 24 h in mouse ovaries and primary GCs. Treatment with SR9009, an agonist of NR1D1, significantly reduced the expression of Bmal1, Per2, and Dbp in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, Nr1d1 knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of Bmal1-/- mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited Atg5 transcription by binding to two putative retinoic acid-related orphan receptor response elements within the promoter. In addition, rapamycin-induced autophagy and ATG5 expression were partially reversed by SR9009 treatment in mouse GCs. Taken together, our current data demonstrated that the circadian clock regulates GC autophagy through NR1D1-mediated inhibition of ATG5 expression, and thus, plays a role in maintaining autophagy homeostasis in GCs.

Spontaneous Recovery of Circadian Organization in Mice Lacking a Core Component of the Molecular Clockwork

Circadian rhythms are generated by interlocked transcriptional-translational feedback loops of circadian clock genes and their protein products. Mice homozygous for a functional deletion in the Period-2 gene ( Per2m/m mice) exhibit short free-running circadian periods and eventually lose behavioral circadian rhythmicity in constant darkness (DD). We investigated Per2m/m mice in DD for several months and identified a categorical sex difference in the dependence on Per2 for maintenance of circadian rhythms. Nearly all female Per2m/m mice became circadian arrhythmic in DD, whereas free-running rhythms persisted in 37% of males. Remarkably, with extended testing, Per2m/m mice did not remain arrhythmic in DD, but after varying intervals spontaneously recovered robust, free-running circadian rhythms, with periods shorter than those expressed prior to arrhythmia. Spontaneous recovery was strikingly sex-biased, occurring in 95% of females and 33% of males. Castration in adulthood resulted in male Per2m/m mice exhibiting female-like levels of arrhythmia in DD, but did not affect spontaneous recovery. The circadian pacemaker of many gonad-intact males, but not females, can persist in DD for long intervals without a functional PER2 protein; their circadian clocks may be in an unstable equilibrium, incapable of sustaining persistent coherent circadian organization, resulting in transient cycles of circadian organization and arrhythmia.

New Insights Into Cancer Chronotherapies

Circadian clocks participate in the coordination of various metabolic and biological activities to maintain homeostasis. Disturbances in the circadian rhythm and cancers are closely related. Circadian clock genes are differentially expressed in many tumors, and accelerate the development and progression of tumors. In addition, tumor tissues exert varying biological activities compared to normal tissues due to resetting of altered rhythms. Thus, chronotherapeutics used for cancer treatment should exploit the timing of circadian rhythms to achieve higher efficacy and mild toxicity. Due to interpatient differences in circadian functions, our findings advocate an individualized precision approach to chronotherapy. Herein, we review the specific association between circadian clocks and cancers. In addition, we focus on chronotherapies in cancers and personalized biomarkers for the development of precision chronotherapy. The understanding of circadian clocks in cancer will provide a rationale for more effective clinical treatment of tumors.

Prognostic values of the core components of the mammalian circadian clock in prostate cancer

Background Prostate cancer (PC) is one of the most common malignancies in males. Extensive and complex connections between circadian rhythm and cancer were found. Nonetheless, in PC, the potential role of the core components of the mammalian circadian clock (CCMCCs) in prognosis prediction has not been fully clarified. Methods We firstly collected 605 patients with PC from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Survival analysis was carried out for each CCMCC. Then, we investigated the prognostic ability of CCMCCs by Cox regression analysis. Independent prognostic signatures were extracted for the establishment of the circadian clock-based risk score model. We explored the predictive performance of the risk score model in the TCGA training cohort and the independent GEO dataset. Finally, the relationships between risk score and clinicopathological parameters, biological processes, and signaling pathways were evaluated. Results The expression levels of CCMCCs were widely correlated with age, tumor status, lymph node status, disease-free survival (DFS), progression-free survival (PFS), and overall survival (OS). Nine circadian clock genes, including CSNK1D, BTRC, CLOCK, CSNK1E, FBXL3, PRKAA2, DBP, NR1D2, and RORB, were identified as vital prognostic factors in PC and were used to construct the circadian clock-based risk score model. For DFS, the area under the 3-year or 5-year receiver operating characteristic curves ranged from 0.728 to 0.821, suggesting better predictive performance. When compared with T3-4N1 stage, PC patients at T2N0 stage might be benefited more from the circadian clock-based risk score model. Furthermore, a high circadian clock-based risk score indicated shorter DFS (p < 0.0001), early progression (p < 0.0001), and higher 5-year death rate (p = 0.007) in PC. The risk score was related to tumor status (p < 0.001), lymph node status (p < 0.001), and ribosome-related biogenesis and pathways. Conclusions The vital roles of circadian clock genes in clinical outcomes were fully depicted. The circadian clock-based risk score model could reflect and predict the prognosis of patients with PC.

Bmal1- and Per2-mediated Regulation of the Osteogenic Differentiation and Proliferation of Mouse BMSCs by Modulating the Wnt/β-catenin Pathway

Bmal1 and Per2 are the core components of the circadian clock genes(CCGs). Bmal1−/− mice exhibited premature aging characterized by osteoporosis and reduced proliferation ability. The same thing occurred in Per2−/− mice, albeit to a less severe degree. However, whether the effects of Bmal1 and Per2 on proliferation and osteogenic differentiation are synergistic or antagonistic remains unclear. To figure this out, we constructed lentiviral and adenoviral vectors to silence or overexpress Bmal1 or Per2 in bone marrow mesenchymal stem cells (BMSCs), and applied MTT, flow cytometry, RT-qPCR, WB, and ChIP-Seq analyses to identify the underlying mechanism. The results showed Bmal1 and Per2 had synergistic effects on the proliferation and differentiation of BMSCs. Furthermore, Bmal1 and Per2 inhibited the Wnt/β-catenin signaling pathway, accompanied by downregulating Rorα expression and upregulating Rev-erbα expression, both of which were also key elements of CCGs. This may be the mechanism by which Bmal1 and Per2 negatively regulate the osteogenic differentiation of BMSCs.

TAMI-44. ASSOCIATION OF CIRCADIAN CLOCK GENE EXPRESSION WITH GLIOMA TUMOR MICROENVIRONMENT AND PATIENT SURVIVAL

Circadian clock genes have been linked to differences in clinical outcomes in cancer, including gliomas. However, these studies have not accounted for established prognostic markers, including mutations in Isocitrate Dehydrogenase (IDH). To study the connection between circadian clock genes and glioma outcomes while accounting for IDH mutational status, we analyzed multiple publicly available gene expression datasets. Unsupervised clustering of 13 clock gene transcriptomic signatures from The Cancer Genome Atlas resulted in four distinct transcriptomic clusters, two clusters were enriched for IDH mutant (Circadian 1-2) and the others for IDH wild-type gliomas (Circadian 3-4). Within these clusters we observed differential prognosis of the patients by Kaplan–Meier analysis (Circadian 1-2, p=0.0001; Circadian 3-4, p=0.0002) suggesting that these transcriptomic circadian subtypes might reflect different disease states. Further analyses using Cox Proportional Hazards Regression showed that lower Period (PER) gene expression was associated with worse prognosis (increasing PER expression HR=0.655, p=0.007) independent of IDH wild-type status (HR=5.312, p&lt; 0.001) and increasing age (HR = 1.04, p&lt; 0.001). Lower PER expression was associated with enrichment of a number of immune signaling pathways. These findings prompted the exploration of the relationship between microenvironment and clock genes using the Ivy GAP dataset to explore tumor location-specific differences and single cell RNA sequencing data from Darmanis (accession: GSE84465) to explore cell-specific differences. Circadian clock genes were found to be differentially expressed across anatomical tumor locations and cell types, including microglia. In ongoing studies we are examining the role of the microenvironment and PER2 expression on tumor growth by disrupting PER2 expression in tumor cells and microglia using IDH mutant and wild-type in vitro models. Clock gene expression is a potential prognostic biomarker in glioma and further studies to elucidate the importance of circadian rhythms in other cell types beyond the tumor are warranted.