Circadian clock control of breast cancer hallmarks: molecular mechanisms and therapeutic implications.

Circadian molecular clock disruption in chronic pulmonary diseases.

The disruption of the circadian clock by frequent shifts in the light–dark cycle, such as shift-work or frequent jet lag, increases the risk of many diseases, including cancer. Experimental disruption of the circadian clock also increases tumor development in mice, although most studies used the strains that are genetically impaired in melatonin synthesis and secretion. Here, we examined the effects of experimental chronic jet lag with 8 h advances of the light–dark cycle every 2 days for 10 days on the central and peripheral clocks of CBA/N mice, the strain with normal profiles of melatonin synthesis and secretion. Mice were exposed to constant darkness after the 10 days of chronic jet lag. In the suprachiasmatic nucleus (SCN), chronic jet lag shifted the temporal expression of most clock genes examined without causing total disturbance of circadian oscillations. In the liver, the temporal patterns of Per1, Bmal1, and Dbp expression were phase-shifted, and Per2 expression was significantly upregulated by chronic jet lag. Further, the expression of cell cycle-related genes, c-Myc and p53 in the liver was significantly activated by the chronic jet lag schedule with a significant positive correlation between Per2 and p53 expression. We determined the plasma concentrations of melatonin and corticosterone as candidate hormonal messengers of chronic jet lag, but their overall levels were not affected by chronic jet lag. Moreover, the expression of the MT1 melatonin and glucocorticoid receptors in the liver was suppressed by chronic jet lag. These data suggest that in CBA/N mice, frequent advances of light–dark cycles modify the phases of central clock in the SCN and disturb the peripheral clock in the liver and apoptotic functions, which may be associated with the suppression of hormone receptors.

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence, or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. We found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. We also demonstrate that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. Our results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo. Editor's evaluation This manuscript will be of broad interest primarily to readers in the field of Chronobiology, but more in general, also Physiology. The reporter construct generated in this study provides a great tool to dissect with cell and tissue specificity the rhythmic transcriptional oscillations orchestrated by circadian clocks in vivo. Here, the authors take full advantage of such a tool showing how neuronal and peripheral clocks might be differentially regulated and possess distinct properties. https://doi.org/10.7554/eLife.77029.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest The daily rhythms in our lives are driven by biological mechanisms called circadian clocks. These biological clocks are protein machines found in almost every cell and organ of the body, in nearly all living things, from fungi and plants to fruit flies and humans. These clocks control 24-hour cycles of gene activity and behaviour, and are kept in-time by so-called ‘master clocks’ in the brain. Ideally, scientists would be able to observe how circadian clocks work in different parts of the brain in a living animal and track changes throughout the day, as the animal performs different behaviours. However, the tools that are currently available to study circadian clocks do not allow this. To overcome this difficulty, Johnstone et al. used fruit flies to develop a new method that allows scientists to measure the oscillations of the circadian clocks in the brain in real time. Circadian clocks are composed of proteins called ‘transcription factors’ that activate different genes throughout the day, producing different proteins at different times. Transcription factors control the activity of genes by binding to DNA sequences called ‘promoters’ and switching the genes regulated by these promoters on or off. Knowing this, Johnstone et al. engineered fruit flies to carry the gene that codes for a protein called luciferase, which emits light, and placed it under the control of the promoter for the period gene, a gene that is regulated by the circadian clock. To prevent all of the cells in the fly from producing luciferase any time the period promoter was active, Johnstone et al. placed a second gene between the promoter and the luciferase gene. This second gene contains ‘stop’ sequences that prevent luciferase from being produced as long as the second gene is present. Importantly, this gene can be genetically removed from specific cells in live flies, so only these cells will produce luciferase. When Johnstone et al. removed the second gene from specific cells in the fly brain that are involved in controlling behaviours related to the circadian clocks, these cells started emitting light in cycles that reproduced the activity of the circadian clocks. Thus, by monitoring how the brightness of luciferase changed throughout the day in these flies, Johnstone et al. were able to reveal how the circadian clocks work in different parts of the fly brain. They found that each clock had slightly different cycling lengths, suggesting that the clocks work differently in different parts of the brain to control behaviour. Interestingly, Johnstone et al. found that if a key gene responsible for communication between cells was mutated, the effects of the mutation also varied in different parts of the brain. This suggests that different clocks respond differently to communication cues. Additionally, the results showed that circadian clock activity also changed with age: older flies had weaker circadian behaviours – fewer changes in both behavioural and genetic activity levels between the day and night – than younger animals. Johnstone et al.’s approach makes it possible to track a living animal’s circadian clocks in different parts of the brain and in different organs in real time without the need to dissect the animal. In the future, this method will help scientists understand the links between different circadian clocks, the genes associated with them, and the behaviours they control. Introduction ‘Circadian rhythms’ collectively refer to ~24 hr oscillations in an animal’s behavior and physiological responses to daily environmental changes. These rhythms are regulated by the circadian clock, a transcription/translation negative feedback loop that controls the ~24 hr oscillations in expression of hundreds of genes in every tissue. Circadian clocks are highly evolutionarily conserved time-keeping machines, from flies to humans. In both organisms, specialized neurons that express circadian clock components are considered the ‘central clock’; circadian clock components in non-neuronal tissue (hereafter, ‘peripheral clocks’) are widely assumed to respond to the central clock (Brown et al., 2019; Franco et al., 2018; Ito and Tomioka, 2016; Patke et al., 2020; Pilorz et al., 2018), likely through secreted factors (Handler and Konopka, 1979). In humans, disruption of the circadian clock is associated with a wide range of pathologies, including neurological, cardiovascular, and metabolic disorders, as well as cancer and aging (Acosta-Rodríguez et al., 2021; Bae et al., 2019; Hood and Amir, 2017a; Hood and Amir, 2017b; Leng et al., 2019; Logan and McClung, 2019; Rana et al., 2020; Shimizu et al., 2016; Sulli et al., 2019; Thosar et al., 2018; Tsuchiya et al., 2020; Zhang et al., 2021). Such a broad variety of pathologies associated with compromised circadian rhythms suggests a need for cheap and effective ways to measure tissue-specific circadian clocks directly in model organisms. In animals, locomotion is the simplest and most rapid way to measure circadian clock output, but this output embodies the cumulative activity of many clocks and does not necessarily represent all clocks equally. Moreover, while ablation of neuronal clocks in flies, mice, and humans leads to loss of sleep/activity rhythms and is thought to cause loss of circadian clock function in many tissues, the hierarchy of dysfunction of tissue-specific clocks during specific disease pathogenesis remains unclear. Currently, individual peripheral clocks can be measured in flies by removing the organ and extracting RNA to assess transcriptional oscillations (Erion et al., 2016; Gill et al., 2015; Litovchenko et al., 2021; Wang et al., 2004; Xu et al., 2011). Such terminal qRT-PCR outputs from explanted organs measure clock function only for that time point in the lifespan of the organism and can be time-, cost-, and labor-intensive. Given that circadian clocks appear to be linked to a wide range of physiologies, including metabolism as well as various behavioral disorders, there is a need to monitor distinct cell- and tissue-specific circadian clocks directly, in vivo, and in real time in Drosophila, similar to reporters developed in mouse models (Sinturel et al., 2021; Smith et al., 2022). We developed a genetically encoded reporter to monitor distinct clocks in Drosophila that we call Locally Activatable BioLuminescence (LABL), offering both high spatial and temporal resolution of clock oscillations in vivo. Our data reveal that tissue-specific clocks have similar but distinct properties of oscillation. To determine if tissue-specific clocks are differentially affected by whole-body mutations, we tested flies lacking a functional PDF (pigment dispersing factor) receptor (han5304, also known as pdfr5304) (Hyun et al., 2005) to demonstrate the properties of clock oscillations LABL can help uncover. PDF is a neuropeptide that elicits a cAMP response from most circadian neurons in the brain and is required to maintain robust rhythmic behaviour in constant dark conditions (Helfrich-Förster, 1995; Helfrich-Förster et al., 2000; Park et al., 2000; Renn et al., 1999; Shafer et al., 2008). While tim01 flies (lacking a functional clock) become completely behaviourally arrythmic and han5304 flies become mostly behaviourally arrhythmic in constant dark conditions, quantification of han5304 fly neuronal clocks reveals infradian oscillations over a narrower range with a mean of ~60 hr, suggesting that loss of different circadian components can disrupt circadian locomotor activity in different ways. When peripheral clocks of a han5304 mutant fly are investigated, they continue to oscillate, but with decreased stability. Here, we demonstrate that LABL reporter flies can be used to measure distinct circadian clocks in different neuronal subpopulations and peripheral tissues in real time and in vivo. The differential changes to distinct clocks caused by the han5304 mutation underscores the assertion that tissue-specific circadian clocks are differentially regulated. We believe that this technology will be critical in the interrogation of distinct circadian clocks in future studies, particularly in monitoring peripheral clock function during disease progression in Drosophila. Results Construction of LABL To monitor distinct clock oscillations in real time, in vivo, we designed a genetically encoded reporter that we call LABL. LABL was constructed into an attB cloning vector for Drosophila embryo injection and PhiC31-mediated genome integration (Bischof et al., 2007; Figure 1—figure supplement 1). LABL is comprised of a per promoter fused to mCherry flanked by FRT sequences, which was subsequently fused to Luc2 (pGL4.10) (Figure 1A). Three stop codons placed 3’ of mCherry are designed to block Luciferase expression. The employed per promoter (~6.7 kb) responds to clock regulation through the CLK/CYC transcription activator complex (Bargiello et al., 1984; Darlington et al., 1998). Tissue-specific expression of Flipase (FLP) triggers recombination at the FRT sites, excising mCherry from the genome and leaving Luciferase under per promoter control. To test the functionality of LABL in vitro, we monitored FLP-driven LABL activity in cultured S2 cells (Figure 1B and C). Since S2 cells do not express CLK, which is required to activate the per promoter, we co-transfected LABL plasmid with Clk-CFP, with and without FLP. These cells were subsequently imaged for CFP and mCherry expression and monitored for luminescence. Cells expressing FLP exhibited increased luminescence and a loss of mCherry fluorescence. Cells lacking FLP exhibited no luminescence but increased mCherry fluorescence. Thus, these data demonstrate that the LABL reporter is functional as designed and can be activated by FLP expression. Figure 1 with 1 supplement see all Download asset Open asset Design and activity of LABL reporter. (A) The Locally Activatable Bioluminescence (LABL) reporter construct. In architecture, the per promoter (pPer) is fused to mCherry followed by luciferase (Luc). The mCherry gene and three stop codons are flanked by FRT recombination sequences. Expression of Flipase (FLP) excises out mCherry (dashed lines), leaving luciferase under period promoter regulation. (B) Fluorescence image of LABL-expressing S2 cells. Expression of LABL reporter with actin promoter-driven Clk (pAc-Clk-CFP), FLP (pAc-FLP), or both reveals CLK-dependent expression of mCherry in the absence of FLP. Scale bar represents 5 μm. (C) LABL can be activated in cultured S2 cells. Lysed S2 cells emit measurable luminescence in a luciferase assay when expressing LABL reporter in a FLP-dependent manner. ****: p < 0.0001. Luminescence oscillations of transcription activity reflect behavioural rhythms Having established its functionality in cultured cells, we proceeded to assess LABL in adult flies. The LABL reporter plasmid was used to generate reporter flies which were then monitored in a luminometer using arenas designed to hold 15 flies on top of fly food supplemented with luciferin (Figure 2A). LABL flies carrying the tim-UAS-Gal4 (TUG) pan-circadian tissue driver were crossed to flies carrying UAS-FLP, and the progeny monitored for luminescence activity in constant darkness (Figure 2B). The raw luminescence data exhibited an oscillating rhythm and a gradual decay, as expected (Brandes et al., 1996; Stanewsky et al., 1997). Figure 2 with 1 supplement see all Download asset Open asset Measurement, quantification, and analysis of luminescence from LABL flies. (A) LABL activation strategy in Drosophila brain. Fly brain schematic illustrates how LABL can be activated using tissue-specific Gal4 drivers which express UAS-FLP2 to excise mCherry out of the genome in some neurons to permit Luciferase expression (yellow circles) under the regulation of the period promoter, leaving other neurons untouched (red circles). Fifteen LABL flies are placed in custom-made plates containing luciferin mixed with standard fly food. Plates are loaded into a luminometer and luminescence from each cohort is recorded for analysis. (B) Raw luminescence measurements of live flies. Photons were detected from four replicates of 15 flies expressing LABL reporter, UAS-FLP2 and tim-UAS-Gal4 over 9 days at 4-min temporal resolution. (C) Comparison between luminescence signal (upper graph) and locomotor activity (lower graph) of wild-type flies. Luminescence signal from flies described in panel B is normalized to exponential decay of signal, the values are averaged into 30 min bins, and the mean of the four experiments presented, +/-SEM (thickness of curve). Light grey and dark grey backgrounds represent subjective day and subjective night, respectively. Vertical solid black lines divide days. The peaks and troughs of the presented curves are represented by dots, and are the mean of four experiments, +/-SEM. Lines connecting the dots of peaks or troughs are best-fit S-curves. The vertical dashed lines define the point of inflection of each S-curve, which is used to define the A50. Locomotion activity is measured in beam breaks (counts) per 30-min bouts. Curve in lower graph represents rhythmic locomotor activity of 25 flies, +/-SEM (thickness of curve). White background indicates lights on. Vertical solid lines divide days. Light grey and dark grey backgrounds represent subjective day and subjective night, respectively. Blue and yellow dots represent peaks of “morning anticipatory” and “evening anticipatory” locomotion activity, respectively. The vertical dashed lines define the point of inflection of each S-curve, fitted to peaks of activity, which is used to define the decay of amplitude of peaks of behavior. (D) Calculation of oscillation peaks and troughs. A representative single replicate from experiment in panels B and C is plotted. Dots represent averaged luminescence signal in 30-min bins. A sinusoidal curve spanning 2 days is fitted to the data in 1 day increments (distinct colored curves). The peaks and troughs of each curve are calculated (triangles), averaged for both x- and y-values and recorded. This process is repeated for all four replicates and the resulting average is reported as shown in panel C. (E) Changes in oscillation period over time determined by Morlet wavelet fitting. Wavelets of different periods were fitted to luminescence signal from a single representative replicate from experiment in panel B and C, and assigned a confidence interval, across time (upper graph). Periods with highest confidence intervals at a time point (i.e. across the x-axis) were plotted as white dots. Confidence intervals of 25% or less were omitted. These values were replotted along with the other replicates (below), with varying shades of grey representing each of the four experiments. The dotted horizontal line denotes 24 hr as a point of reference. Right panel: All data points without the time dimension are plotted. Bar represents the mean, +/-SD. We next compared pan-circadian tissue luminescence oscillations with behavioural rhythms. The recorded luminescence activity was normalized to the gradual decay in signal and plotted as an average of four experiments (Figure 2C, top panel). Control flies lacking a driver exhibited no discernable oscillation (white line). To characterize the luminescence oscillations, we first quantified the decay in amplitude (the difference in y-value, between peaks and troughs) of signal. Data points were binned into 30-min time intervals and a 48-hr sinusoidal curve was fitted to the data at 24-hr intervals (Figure 2D). The coordinates of the local minima and maxima were recorded, averaged, and plotted over the decay-normalized luminescence signal to reveal the amplitude of oscillation across time (black circles) (Figure 2C). An S-curve fitted to the changing local minima and maxima (black line) revealed points of inflection coinciding with Day 6 of constant darkness (vertical dashed lines). We used this point of inflection, which is the time at which the amplitude falls to 50% between maximum and minimum (A50), as a measure of clock stability because decay of oscillations into arrhythmic transcription may exceed the timeline of this assay. Genotypically identical flies were measured for locomotor activity and their behavioral rhythms plotted (Figure 2C, bottom panel). We found that the peaks of morning anticipation (yellow circles) decayed rapidly, allowing the evening anticipation peaks (blue circles) to dominate behavioral oscillations in constant dark conditions. Focusing on the change in evening anticipation peaks, we found that a fitted S-curve revealed a point of inflection (A50) at ~day 6, coincident to the A50 observed in luminescence oscillations. We conclude that the decay of amplitude of oscillation of the ubiquitously expressed molecular clock and behavioural rhythms are consistent with each other. We next characterized the change of period of luminescence oscillation across time. A Morlet wavelet was fitted onto the measured luminescence oscillations (Figure 2E). Period values with highest confidence intervals revealed a steady ~23.5 hr period of oscillation across time; specifically, oscillations occurred with an average period of 23.57 hr over nine days in constant darkness. This oscillation period also mirrored the behavioural period, with luminescence oscillation and behavioural period statistically the same, at ~23.5 hr. Thus, luminescence be detected in flies in which LABL was activated using and the pan-circadian Gal4 driver and revealed between transcription oscillations and behavioral rhythms of the LABL signal is to ubiquitously expressed luciferase reporters and is clock To determine the of LABL as a reporter, we compared LABL oscillations to ubiquitously expressed luciferase To this we compared our data to data from promoter fused to (Brandes et al., and promoter and of the per gene fused to et al., flies, other reporter (Figure Since Gal4 expression in all clock cells, we expected LABL luminescence signal to be to luminescence signal. the oscillation of luminescence signal was in period, and (Figure The on the other a of the and there is a between per transcription and per and Stanewsky et al., we expected a difference between LABL luminescence signal and signal. luminescence signal was compared to both LABL and flies et al., 1997). Thus, LABL is to both and flies. Figure with 1 supplement see all Download asset Open asset LABL oscillations are to other luminescence reporters and respond to circadian clock (A) Comparison of luminescence LABL activated by tim-UAS-Gal4 (black is plotted per promoter fused to luciferase and curve). (B) LABL is activated by tim-UAS-Gal4 in a tim01 genetic graph represents locomotor Locomotion activity is measured in beam breaks (counts) per 30-min bouts. Curve represents rhythmic locomotor activity of 25 flies, +/-SEM (thickness of curve). White background indicates lights on. Vertical solid lines divide days. Light grey and dark grey backgrounds represent subjective day and subjective night, respectively. graph illustrates luminescence signal over time. Luminescence signal is normalized to exponential decay of signal, values averaged into 30-min bins, as the mean of four experiments presented, +/-SEM (thickness of curve). Light grey and dark grey backgrounds represent subjective day and subjective night, respectively. Vertical solid black lines divide days. graph illustrates changes in oscillation period over time determined by Morlet wavelet fitting. are as described in Figure represents an replicate of wavelet period in the tim01 genetic is a of period of each of the four plotted. replicates are background represents range of of the han5304 genetic (C) LABL is activated by tim-UAS-Gal4 in a han5304 genetic are as described in panel of period in the infradian oscillation range – is different in han5304 flies when compared to tim01 flies, determined by the test p < the clock behavioural and loss of transcriptional oscillation. To that the transcription oscillations we observed using LABL were we monitored LABL luminescence oscillations in a genetic background lacking functional expression (Figure both locomotor activity and transcription oscillation of tim01 flies were arrhythmic and panel). Importantly, our to Morlet to the luminescence data revealed periods from hr to hr, consistent with circadian panel). of PDF using han5304 mutant flies PDF receptor lacking its and rhythmic behavior in the first days of constant but results in arrhythmic behavior in a of flies (Hyun et al., When we characterized LABL oscillations in a han5304 background (Figure we found that the of flies arrhythmic in their locomotor activity in the second day of constant as While there was also a rapid decay in transcription measured by revealed that ~24 hr oscillations decayed into a infradian oscillations with a narrower range of Such a decay from an ~24 hr oscillation and confidence wavelet infradian oscillation of luminescence was not in flies lacking a functional clock (i.e. tim01 A test reveals that the period the hr range of the han5304 mutant flies was different than that of tim01 flies (Figure and C, of the of infradian rhythms in we to determine if we observe these oscillations using other by locomotion assay or We first measured locomotion rhythms of wild-type and tim01 flies, then a analysis for behavioural period (Figure supplement 1A). In the hr wild-type flies robust ~24 hr rhythms with high of han5304 flies behavioural periods that 24 hr with a wide and a of and tim01 flies no discernable of behavioural period and a of We repeated the analysis for the hr range and found of behavioural periods at hr and hr for both and han5304 flies. tim01 flies showed no All flies had an of to the hr and hr we observed in wild-type and han5304 flies be considered infradian to the of that was to that of tim01 flies, we do not these data of infradian oscillations. We next protein from han5304 fly then for and by to determine if we observe infradian oscillations (Figure supplement We used protein from because protein from flies not oscillating protein signal by (Figure supplement to protein in in flies We observed and oscillations from from both wild-type and the first day of han5304 flies in constant darkness. However, the and not represent oscillations to a hr period in these We conclude that the amplitude of luminescence oscillation may be to observe by locomotion or Circadian drivers used to activate LABL reveal oscillating luminescence Since the driver can be used to activate LABL in flies to oscillating luminescence signal, we to demonstrate that other used circadian drivers can also be used as We FLP expression using and (Figure and Figure supplement 1). We found that the oscillation with the signal from flies in which LABL was activated by and and this well with the of neurons that were for LABL activation when compared with LABL (Figure and drivers oscillations with than would be expected in to their neurons (Figure supplement suggesting that neurons or tissues may be periods were at ~24 hr. We conclude that LABL can also be used in with more tissue-specific Figure with 2 see all Download asset Open asset Luminescence oscillations of distinct clocks measured in and flies. Luminescence signal using different Gal4 drivers to activate LABL compared in day and day flies. used to activate LABL are or in wild-type flies. Luminescence values are normalized and averaged into 30 bins, and the mean of four experiments presented, +/-SEM (thickness of curve). Light grey and dark grey backgrounds represent subjective day and subjective night, respectively. Vertical solid black lines divide days. The peaks and troughs of the presented curves are represented by dots, which are the mean of four experiments, +/-SEM. dots are no be Lines connecting peaks or troughs are best-fit S-curves. The vertical dashed lines define the point of inflection of each S-curve, which is used to define the A50. a vertical dashed line is no S-curve be or no decay be flies exhibit rapid loss of amplitude of luminescence oscillation and of infradian oscillations Since luminescence oscillations in han5304 exhibited differences in

In most organisms ranging from cyanobacteria to humans, the endogenous timekeeping system temporally coordinates the behavioral, physiological, and metabolic processes with a periodicity close to 24 h. The timing of these daily rhythms is orchestrated by the synchronized oscillations of both the central pacemaker in the brain and the peripheral clocks located across multiple organs and tissues. A growing body of evidence suggests that the central circadian clock and peripheral clocks residing in the metabolically active tissues are incredibly well coordinated to confer coherent metabolic homeostasis. The interplay between nutrient metabolism and circadian rhythms can occur at various levels supported by the molecular clock network, multiple systemic mechanisms, and the neuroendocrine signaling pathways. While studies suggest the reciprocal regulation between circadian clock and metabolism, it is important to understand the precise mechanisms and the underlying pathways involved in the cross-talk among circadian oscillators and diverse metabolic networks. In addition to the internal synchronization of the metabolic rhythms, feeding time is considered as a potential external synchronization cue that fine tunes the timing of the circadian rhythms in metabolic peripheral clocks. A deeper understanding of how the timing of food intake and the diet composition drive the tissue-specific metabolic rhythms across the body is concomitantly important to develop novel therapeutic strategies for the metabolic disorders arising from circadian misalignment. This review summarizes the recent advancements in the circadian clock regulation of nutrient metabolism and discusses the current understanding of the metabolic feedback signals that link energy metabolism with the circadian clock.

We used time-restricted feeding (TRF) to investigate whether microbial metabolites and the hunger hormone ghrelin can become the dominant entraining factor during chronic jetlag to prevent disruption of the master and peripheral clocks, in order to promote health. Therefore, hypothalamic clock gene and Agrp/Npy mRNA expression were measured in mice that were either chronically jetlagged and fed ad libitum, jetlagged and fed a TRF diet, or not jetlagged and fed a TRF diet. Fecal short-chain fatty acid (SCFA) concentrations, plasma ghrelin and corticosterone levels, and colonic clock gene mRNA expression were measured. Preventing the disruption of the food intake pattern during chronic jetlag using TRF restored the rhythmicity in hypothalamic clock gene mRNA expression of Reverbα but not of Arntl. TRF countered the changes in plasma ghrelin levels and in hypothalamic Npy mRNA expression induced by chronic jetlag, thereby reestablishing the food intake pattern. Increase in body mass induced by chronic jetlag was prevented. Alterations in diurnal fluctuations in fecal SCFAs during chronic jetlag were prevented thereby re-entraining the rhythmic expression of peripheral clock genes. In conclusion, TRF during chronodisruption re-entrains the rhythms in clock gene expression and signals from the gut that regulate food intake to normalize body homeostasis.

The circadian clock is a tightly regulated mechanism that has evolved in most organisms to enable them to anticipate daily reoccurring changes in the environment. Expression analysis in mouse suggests that about 10% of the mammalian transcriptome may be under circadian control, however the overlap between different tissues is very small [1]. Mechanisms determining this cell type specificity of clock controlled genes are not yet understood. The human osteosarcoma cell line U2OS expresses a functional circadian clock and has been used in many circadian studies [2, 3, 4], yet, except for components of the core clock machinery, no genes have been found to be rhythmically expressed in this cell line in a microarray analysis [3] whereas about 3000 rhythmic transcripts have been identified in mouse liver [3, 5, 6]. This work shows ChIP-sequencing data of BMAL1, CLOCK, and CRY1 that indicates that the circadian transcription regulators bind in sequence-specific manner to several thousand sites in the genome of U2OS, comparable to the number found in mouse liver [5]. However, time course microarray experiments carried out to verify functionality of these sites, found only 58 rhythmic genes harboring a binding site, suggesting a high amount of opportunistic binding that cannot be linked to transcription. Even most genes with high-scoring binding sites in their promoter were apparently arrhythmic. U2OS cell lines stably expressing luciferase reporter constructs under control of representative promoters were generated and revealed that BMAL1 and CLOCK functionally act on these, but rhythmic expression appears to be superimposed by a high basal transcription. The data indicates that high amplitude transcription rhythms are masked by constitutive transcription that overwrites the rhythmic contribution of the circadian clock, resulting in apparent arrhythmicity due to low amplitude oscillations. This work suggests that the cell-type specific circadian transcriptome is not determined on the level of BMAL1-CLOCK binding and rhythmic activation of transcription by these circadian regulators, but rather by the cell-type specific ratio of circadian versus genespecific or general transcription regulators. Bibliography: [1] Satchidananda Panda, Marina P. Antoch, Brooke H. Miller, Andrew I. Su, Andrew B. Schook, Marty Straume, Peter G. Schultz, Steve A. Kay, Joseph S. Takahashi, and John B. Hogenesch. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell, 109(3):307–20, May 2002. [2] Christopher Vollmers, Satchidananda Panda, and Luciano DiTacchio. A high-throughput assay for siRNA-based circadian screens in human U2OS cells. PloS ONE, 3(10):e3457, January 2008. [3] Michael E. Hughes, Luciano DiTacchio, Kevin R. Hayes, Christopher Vollmers, S. Pulivarthy, Julie E. Baggs, Satchidananda Panda, and John B. Hogenesch. Harmonics of circadian gene transcription in mammals. PLoS Genetics, 5(4):e1000442, April 2009. [4] Bert Maier, Sabrina Wendt, Jens T. Vanselow, Thomas Wallach, Silke Reischl, Stefanie Oehmke, Andreas Schlosser, and Achim Kramer. A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock. Genes & Development, 23(6):708–18, March 2009. [5] Guillaume Rey, Francois Cesbron, Jacques Rougemont, Hans Reinke, Michael Brunner, and Felix Naef. Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver. PLoS Biology, 9(2):e1000595, February 2011. [6] Nobuya Koike, Seung-Hee Yoo, Hung-Chung Huang, Vivek Kumar, Choogon Lee, Tae-Kyung Kim, and Joseph S. Takahashi. Transcriptional Architecture and Chromatin Landscape of the Core Circadian Clock in Mammals. Science, 338(6105):349–54, August 2012.

Purpose: For breast cancer (BC) patients treated with adjuvant chemotherapy (CT), the optimal time to initiation of adjuvant radiotherapy (TTR) from definitive surgery is still controversial especially with modern systemic therapy, while the impact of TTR from completion of CT has not been reported to date. The current study aims to evaluate the impact of TTR from surgery and from completion of CT on survival outcomes in non-metastatic BC patients according to BC subtype. Methods and Materials: BC patients who were treated with definitive surgery followed by adjuvant CT and received adjuvant radiotherapy (RT) from January 2009 through December 2015 in a single institution were included in this study. Patients receiving neoadjuvant therapy were not enrolled. According to our clinical practice, if adjuvant treatments were well organized, RT could be initiated within 180 days following surgery and with completion of most up-to-date CT regimens. As a result, patients were categorized into two groups according to TTR from surgery as ≤180 and &amp;gt;180 days and according to TTR from CT as ≤12 and &amp;gt;12 weeks. The survival curves were estimated by the Kaplan-Meier method and compared by log-rank test. The independent effect of TTR from surgery and TTR from CT were separately tested using a Cox proportional hazards model for multivariate analysis after adjusting for these variables that were statistically significant on univariate analysis. Results: In total, 989 patients were enrolled. The number of patients with HR-positive, triple-negative (TN) and HER2-positive BC was 590, 196, and 203, respectively. The median follow-up was 43 (range: 4 to 117) months. The median TTR from surgery was 180 (range: 24 to 117) days and from completion of CT was 29 (range: 7 to 247) days. The 5-year recurrence-free survival (RFS), locoregional RFS (LRRFS), distant RFS (DRFS) and overall survival (OS) were 88.0%, 96.9%, 89.3% and 93.5%, respectively. The 5-year OS was 94.7%, 88.3% and 95.2% in patients with HR-positive, TN and HER-2 positive BC, respectively (P&amp;lt;0.01). Initiation of RT &amp;gt;12 weeks after completion of CT was associated with worse OS (5-year 94.0% vs 85.1%, p=0.006), &amp;gt;180 days after surgery was associated with worse DRFS (5-y 91.7% vs 86.9%, p=0.004) and worse RFS (5-y 90.7% vs 85.4%, p=0.003). In the multivariable analysis, TTR after completion of CT remained independent prognostic factor for OS (hazard ratio [HR], 2.81; 95% CI, 1.17 to 6.74; P=0.02) and TTR &amp;gt;180 days after surgery was also significantly associated with worse DRFS (HR, 1.79; 95% CI, 1.09 to 2.93; P=0.02) and RFS (HR, 1.71; 95% CI, 1.09 to 2.69; P=0.02). In patients with HR-positive BC, TTR &amp;gt;12 weeks after completion of CT was significantly associated with worse OS, while &amp;gt;180 days after surgery was with adverse DRFS (5-y 91.0% vs. 83.8%, p=0.004), RFS (5-y 89.8% vs. 82.8%, p=0.003), and OS (5-y96.6% vs 91.8%, p=0.026). However, these associations between TTRs and survival outcomes were not found in patients with HER2-positive BC or TNBC. Conclusion: In BC patients indicated for CT, delaying initiation of RT after definitive surgery or after completion of CT both adversely impact on survival outcomes. Efforts should be made to minimize delays in the initiation of RT in the above two TTR settings. Citation Format: Lu Cao, Jia-Yi Chen. Impact of delaying initiation of RT following definitive surgery or following adjuvant chemotherapy on survival outcomes in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-12-19.

Preface

Drosophila Ebony Activity Is Required in Glia for the Circadian Regulation of Locomotor Activity

BackgroundThe circadian clock integrates external environmental changes into the internal physiology of organisms. Perturbed circadian clocks due to misaligned light cycles increase the risk of diseases, including metabolic disorders. However, the effects of sex differences in this context remain unclear.MethodsCircadian misalignment was induced by a chronic jet lag (CJL) shift schedule (light-on time advanced by 6 h every 2 days) in C57BL/6N male and female mice. Core body temperature and activity rhythms were recorded using a nano tag, and the gene expression rhythms of clock and clock-controlled genes in the liver and adrenal glands were analyzed using qPCR. Glucose metabolism and insulin response were evaluated using glucose tolerance, insulin sensitivity, and glucose response assays. Castration and testosterone replacement were performed to assess the fundamental role of testosterone in male phenotypes under CJL.ResultsUnder CJL treatment, male mice exhibited increased weight gain, whereas females exhibited decreased weight gain compared to that of the respective controls. CJL treatment induced a lower robustness of circadian rhythms in core body temperature and a weaker rhythm of clock gene expression in the liver and adrenal glands in females, but not in males. Only male mice exhibited glucose intolerance under CJL conditions, without the development of insulin resistance. Castrated mice without testosterone exhibited decreased weight gain and reduced robustness of body temperature rhythm, as observed in intact females. Testosterone replacement in castrated mice recovered the CJL-induced weight gain, robustness of temperature rhythm, and glucose intolerance observed in intact males.ConclusionsSignificant sex-based differences were observed in circadian clock organization and metabolism under CJL. Testosterone plays a crucial role in maintaining the circadian clock and regulating CJL metabolism in males.

Sarcopenia is correlated with poor clinical outcomes in breast cancer (BC) patients. However, there is no precise quantitative study on the correlation between body composition changes and BC metastasis and survival. The present study proposed a deep learning radiomics (DLR) approach to investigate the effects of muscle and fat on distant metastasis and death outcomes in BC patients. Image feature extraction was performed on 4th thoracic vertebra (T4) and 11th thoracic vertebra (T11) on computed tomography (CT) image levels by DLR, and image features were combined with clinical information to predict distant metastasis in BC patients. Clinical information combined with DLR significantly predicted distant metastasis in BC patients. In the test cohort, the area under the curve of model performance on clinical information combined with DLR was 0.960 (95% CI: 0.942-0.979, P < 0.001). The patients with distant metastases had a lower pectoral muscle index in T4 (PMI/T4) than in patients without metastases. PMI/T4 and visceral fat tissue area in T11 (VFA/T11) were independent prognostic factors for the overall survival in BC patients. The pectoralis muscle area in T4 (PMA/T4) and PMI/T4 is an independent prognostic factor for distant metastasis-free survival in BC patients. The current study further confirmed that muscle/fat of T4 and T11 levels have a significant effect on the distant metastasis of BC. Appending the network features of T4 and T11 to the model significantly enhances the prediction performance of distant metastasis of BC, providing a valuable biomarker for the early treatment of BC patients.

Several studies have recently indicated the activation of the immune system against tumor cells as well as the targeting of cancer cell DNA damage repair mechanisms as effective strategies to target tumor growth. STING is a well-known DNA sensor of innate immunity mostly characterized as a transmembrane protein of various cytoplasmic organelles that senses cytosolic DNA as a danger signal and triggers inflammatory responses. A current cancer immunotherapy strategy relies on the use of STING agonists to boost the patient's immune system through a cytokine-mediated recruitment of immune cells that infiltrate and kill tumor cells. However, the role of the STING pathway in cancer is far to be fully understood as there is otherwise accumulating evidence that activation of the cGAS-STING pathway can have a deleterious outcome. We recently showed that genotoxic treatment of breast cancer PDXs and cell lines triggered the STING pathway. Genetic inhibition of this pathway in MCF7 cells increased genotoxic treatment efficacy by promoting cell death and delaying cell colony regrowth, indicating that STING pathway intrinsically promotes cell resistance to treatment. In this study, we show that STING silencing decreased cell viability in a panel of classical or PDX-derived breast cancer cell lines irrespective of their ER status and of the genotoxic treatment received. Cell fractionation indicates that part of the STING pool intrinsically resides in the nucleus of various malignant and non-malignant cells. Fluorescence and electron microscopy show that STING partly resides at the inner membrane of the nucleus, and mass-spectrometry analysis revealed that STING interacts with core proteins of the non-homologous end joining (NHEJ) DNA damage repair (DDR) complex. STING promotes NEHJ-related protein assembly with chromatin, and its silencing decreases DDR and cell viability, while STING overexpression protects cancer cells from genotoxic treatment. STING involvement in DDR is independent of the classical STING-TBK1-IFN inflammatory response, thus identifying a new functional pathway for STING. STING nuclear localization was confirmed in a panel of breast cancer patient-derived xenografts and in surgical samples from breast cancer patients that received neoadjuvant chemotherapy. Evaluation of the impact of STING expression on patient outcome via the Kaplan Meier plotter show that overall STING expression level is positively correlated with favorable outcome in breast cancer patients, however high STING expression in breast and ovarian cancer patients treated with adjuvant chemotherapy is associated with poor prognosis. These findings place STING at the crossroad of DDR and immune surveillance, two major pathways for tumorigenesis and tumor survival. Citation Format: Laura Cheradame, Ida Chiara Guerrera, Julie Gaston, Alain Schmitt, Vincent Jung, Marion Pouillard, Nina Radosevic-Robin, Mauro Modesti, Jean-Gabriel Judde, Vincent Goffin, Stefano Cairo. A non-canonical, cell-autonomous STING function protects breast cancer cells from intrinsic and genotoxic-induced DNA instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2038.

In mammals, circadian rhythms in peripheral organs are impaired when animals are maintained in abnormal environmental light-dark cycles such as constant light (LL). This conclusion is based on averaged data from groups of experimental animals sacrificed at each time point. To investigate the effect of LL housing on the peripheral clocks of individual mice, an in vivo imaging system was used to observe the circadian bioluminescence rhythm in peripheral tissues of the liver, kidney, and submandibular salivary gland in PER2::LUCIFERASE knock-in mice. Using this technique, we demonstrated that the majority of individual peripheral tissues still had rhythmic oscillations of their circadian clocks in LL conditions. However, LL housing caused decreased amplitudes and a broad distribution of peak phases in PER2::LUCIFERASE oscillations irrespective of the state of the animals' behavioral rhythmicity. Because both scheduled feeding and scheduled exercise are effective recovery stimuli for circadian clock deficits, we examined whether scheduled feeding or scheduled exercise could reverse this impairment. The results showed that scheduled feeding or exercise could not restore the amplitude of peripheral clocks in LL. On the other hand, the LL-induced broad phase distribution was reversed, and peak phases were entrained to a specific time point by scheduled feeding but only slightly by scheduled exercise. The present results demonstrate that LL housing impairs peripheral circadian clock oscillations by altering both amplitude and phase in individual mice. The broad distribution of clock phases was clearly reversed by scheduled feeding, suggesting the importance of scheduled feeding as an entraining stimulus for impaired peripheral clocks.

The circadian clock is an endogenous, self-sustained biological rhythm. Different organisms possess different period of the circadian clock, ranging from 20 to 28 h. Synchronizations of the circadian clock contribute to the organisms’ fitness. This synchrony refers not only to the synchronization between the body clock and the environment, but also to that between the central and peripheral clocks. The synchronization is a process that the organism’s circadian clock is reset by the outside environment. Being synchronized often makes the organism oscillate in a period of 24 h, that of the earth’s rotation. More importantly, being synchronized changes the phase of the organism’s clock, for example activity onset, to a stable phase relationship to the environmental time cues, like the sunrise. Pittendrigh had raised the phase response curve (PRC) model to explain how light synchronizes the circadian clock by phase shifting the clock. His PRC model successfully predicts entrainment on the basis of period changes. Light pulse is considered as the most effective zeitgeber that can synchronize an organism’s circadian clock. Phase response curves show distinct similarities from diverse organisms, including both diurnal and nocturnal animals. Better understanding on these synchronizations are built on recent advances in the following aspects, the molecular feedback loops, the novel photoreceptor melanopsin and its expressing cells, and the hierarchical structures constituting the mammalian clock system. This review summarizes recent progresses on topics of the synchronizations of both the body clock and the environment, and the central and peripheral clocks. It discusses how light and food cues mediate the synchronization of the clock system, followed with introducing main human disorders in which desynchronized body clocks are involved. Light pulse transmits the environmental time cues to the suprachiasmatic nucleus (SCN) through the retino-hypothalamic tract (RHT). SCN is located in the hypothalamus and serves as the central oscillator in mammals. Food is another important zeitgeber that synchronizes the circadian clock. Feeding behavior passes the timing information to the central oscillator, SCN, by taking advantages of the circulating hormones and metabolites. After receiving information from either light or food, SCN integrates the information and passes them along to peripheral clocks that locate in different organs, finally reaching to a coherent circadian system for the entire multicellular organism. Therefore, this review offers basic knowledge to further researches on the clock synchronization, and expects people understand that keeping our body clocks in synchrony is important to our health.

A new role of TRPM8 in circadian rhythm and molecular clock.