Circadian clock, carcinogenesis, chronochemotherapy connections

Abstract

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.

Keywords: XR-seq; cisplatin; colorectal cancer; cryptochrome; nucleotide excision repair; transcription–translation feedback loop; xenografts.

Figure 1

Molecular mechanism of the mammalian circadian clock.A, model for circadian entrainment by light. The “master” clock in the suprachiasmatic nucleus (SCN) in the brain is entrained by neural input from photoreceptors in the retina. The master clock in turn maintains a coherent rhythmicity among clocks in peripheral tissue cells via neural signals and humoral factors. B, the positive (CLOCK-BMAL1) and negative (CRY-PER-CK1δ) arms of the TTFL are in two separate complexes. Mouse liver nuclei were harvested at ZT19 and the extract was separated by glycerol gradient velocity sedimentation along with reference proteins (thyroglobulin [669 kDa, 19S], β-amylase [222 kDa, 8.9S], and ovalbumin [43 kDa, 3.6S]). Fractions were probed by western blotting using appropriate antibodies. Left panel, western blot; right panel, quantitative scan of the western blot. CLOCK-BMAL1 sediments as a heterodimer (M_r ∼200 kDa), and PER2-CRY1- CK1δ sediments as a larger complex of M_r ∼500 kDa. C, TTFL model for the mammalian clock. The CLOCK–BMAL1 transcriptional activator binds to E-boxes at subjective dawn. At this time CRY1 is abundant and binds to the CLOCK-BMAL1-E-box complex and inhibits transcription (“Blocking type repression”). During the daytime, CRYs are degraded and CLOCK-BMAL1 activates transcription of target genes including Cry and Per. When CRY and PER accumulate, they enter the nucleus in the form of a CRY-PER-CK1δ complex, which transiently interacts with CLOCK-BMAL1-E-box (illustrated by brackets), phosphorylates CLOCK, and causes dissociation of the activator heterodimer (“Displacement type repression”). D, clock protein levels in mouse liver over the course of a circadian cycle. The levels are illustrated in the form of qualitative heatmaps, and the consequence of this clock protein change on clock-controlled Nr1d1 and Dbp gene transcription over the course of the day is plotted. Adapted with permission from Cao et al. (25).

Figure 2

Meta analysis studies on circadian disruption and cancer incidence. The studies analyzed by Dun et al. (34) met the following criteria: (1) night-shift work was reported; (2) cancer risk was investigated; (3) cohort studies, case-control studies, or nested case-control studies; (4) the risk was estimated by odds ratio (OR), risk ratio, or hazard ratio, with 95% confidence interval (CI). Cancer risks among individuals with different classifications of night work duration (0–5, 6–10, 11–15, 16–20, 21–25, and ≥26 years) are plotted. Taking all eligible studies together, night-shift work did not increase the risk of cancer in any group of night workers. Image modified with permission from Ref.34 and used under Creative Commons.

Figure 3

Genetically modified model animal studies.A, Kaplan–Meier plots of death from cancer from two different studies of mice with clock gene mutations (31, 41). Eight-week-old mice of the indicated genotypes were exposed to 4 Gy of IR at ZT10 and observed for 80 weeks (B) Effect of Cry mutation on cancer incidence and mortality in mouse strains with a predisposition to cancer. Kaplan–Meier plots of death from cancer are shown. Left, p53^−/− (red) and p53^−/−;Cry1/2^–/– (green and blue) survival probabilities. Data shown by the green line have been published (42), and the unpublished data shown by the blue line were obtained by a different member of the lab in a blind experimental design (31). (Right) Tumor-free survival of ink4a^–/–;ras(V12G) (blue) and ink4a^–/–;ras(V12G);Cry1/2^–/– (red) mice. The experiment was conducted in male mice maintained under standard conditions of 12 h light–12 h dark cycles and monitored regularly for the appearance of melanomas. There is no statistically significant difference between the two survival curves (p = 0.2), and hence, it is concluded that in this genetic background Cry mutation has no mitigating effect on cancer incidence or progression. Adapted with permission from Sancar et al. (31).

Figure 4

Regulation of the clock by c-MYC and of c-MYC by the clock.A, c-MYC regulates Bmal1 by two mechanisms (55, 56, 57, 58). First, c-MYC, in the form of c-MYC-MAX-MIZ1 heterotrimer directly binds to the MIZ-binding site upstream of the Bmal1 promoter and directly inhibits its transcription. Second, in the form of c-MYC-MAX it binds to the E-boxes of the REV-ERB α/β genes (Nr1d1/2) and stimulates their transcription. NR1D1/2, in turn, binds to the RORE element of BMal1 and inhibits its transcription. B, regulation of c-MYC at the transcriptional level by the clock (59). The β-Catenin gene (Ctnnb1) contains an E-box in its 35th intron, to which BMAL1-CLOCK bind and act as a context-dependent repressor (23, 24, 59) to interfere with the transcription of Ctnnb1 (top). CRY-PER remove CLOCK-BMAL1 from the intron, activating Ctnnb1 transcription (bottom). β-catenin makes a complex with TCF/LEF, which stimulates c-Myc transcription. In CRY mutants, BMAL1-CLOCK remains bound to the E-box of Ctnnb1 intron and inhibits its transcription, and in the absence of, or with reduced levels of β-Catenin, c-Myc transcription is downregulated (top). C, regulation of MYC by the clock at a posttranscriptional level (60). When CRY2 is overexpressed by a strong promoter, such as the Igu promoter, it interacts with c-MYC and targets it for degradation by the ubiquitin/proteasome pathway, leading to reduced c-MYC levels.

Figure 5

Mechanism of nucleotide excision repair and its control by the circadian clock.A, molecular mechanism of mammalian Global and Transcription-Coupled Repair. Transcribed strand (TS) repair is predominantly determined by the phase of transcription of a given gene. The nontranscribed strand (NTS) repair is controlled by the repair enzyme complex oscillation, which is dictated by XPA damage recognition protein with a maximum at ZT10 for all genes regardless of the phase of transcription. B, schematic of the core clock that controls XPA expression. C, repair patterns of various gene's TS and NTS repair depending on whether they are constitutively expressed or controlled by the circadian clock, their phase of expression, and level of expression.

Figure 6

DNA damage, repair, gene expression, and epigenomic markers for Per1.Per1 is significantly upregulated after cisplatin treatment across all four organs. RNA-seq plus and minus cisplatin (cisp. and cont., respectively) is shown in gray at the top for each organ. Damage-seq and XR-seq data are shown for both strands. Pt-d(GpG) damage (Damage-seq) and repair (XR-seq) distribution on the TS and NTS are shown with − and +, respectively. Epigenetic data from ChIP-seq of H3K4me3 and H3K27me3, as well as DNase-seq, are plotted at the bottom of each organ. We show that the transcriptional and epigenomic profiles of Per1 and neighboring regions across all four organs recapitulate the differences in DNA damage and repair between the TS and NTS. Adapted with permission from Yimit et al. (102).

Figure 7

Transcriptional and circadian control of excision repair of cisplatin-DNA adducts in mice.A, schematic of circadian repair experiment. Mice kept under 12-h light:12-h dark (LD 12:12) conditions were administered cisplatin at the indicated time points, and tissues were harvested 2 h later; the excision products were isolated from the liver and kidney and analyzed by XR-seq. ZT indicates circadian time where ZT0 is light-on and ZT12 is light-off. For each time point three mice were killed for XR-seq. B, genome-wide analysis of TS and NTS repair shows strong preference for TS repair in promoter-proximal regions, throughout gene bodies, and into the transcriptional end site (TES). Preferential repair reversal upstream of the transcription start sites (TSS) is due to bidirectional promoters for most mammalian genes such that the NTS in the gene body becomes the TS upstream of the TSS. The y axis shows reads per kilobase pair per million total reads (RPKM) for 100-nt windows. C, illustration showing the effect of transcription and the combined effects of the circadian clock and transcription on cisplatin repair. Repair patterns of a 295-kb region of chromosome 1 encompassing the Npas2 clock gene and two neighboring genes are shown. Blue, plus strand XR-seq repair reads; red, minus strand XR-seq repair reads. The Npas2 gene is itself clock-regulated and repair of its TS peaks at ZT20-ZT0 and troughs at ZT08. The clock output gene Rpl31 exhibits much weaker rhythmicity in repair that is delayed compared with Npas2 (peak ZT0-ZT08, minimum ZT16). Tbc1d8 exhibits high amplitude and constant TS repair over the entire course of the circadian cycle. Adapted with permission from Yang et al. (103).

Figure 8

Two interdependent circadian programs control repair of the TS and NTS.A, heatmaps of circadian repair cycles of the transcribed strand (TS) and nontranscribed strand (NTS) of 1661 highly rhythmic genes in mouse kidney. Exp/Med is, for each gene, RPKM at a given ZT divided by the median ZT RPKM value. Note the distribution of the repair maxima over the entire circadian cycle for the TS and the single maximum for repair of the NTS due to the circadian-controlled peak repair activity, which manifests itself on the NTS but its contribution to the TS repair is obscured by the much stronger effect of transcription on repair. The scale for selecting the significant cyclical genes is meta2d_pvalue <0.05, meta2d_rAMP >0.1. Each horizontal line represents one gene every 4 h from ZT0 to ZT24 with two replicates. B, radial diagram representation of TS and NTS repair. The TS repair exhibits two peaks corresponding to predawn and predusk, in agreement with numerous transcriptional analyses studies. The NTS repair exhibits a single peak at ZT08-11 in agreement with the peak transcription-independent excision repair activity. The scale for selecting the significant cyclical genes both in TS and NTS is meta2d_pvalue < 0.05, meta2d_rAMP > 0.1. C, examples of dissonance of the TS versus NTS repair. The dissonance is most apparent when the transcription/repair phase is farthest from ZT08, which represents the total repair activity and hence maximum NTS repair. We used three animals per time point for analysis and performed two biological replicates. The time range is from ZT0 to ZT24. In C (as in A), data for ZT0 to ZT24 replicate one are followed by data for ZT0 to ZT24, replicate two. Adapted with permission from Yang et al. (103).

Figure 9

XR-seq analysis of repair in circadian-controlled genes. The screenshot shows repair profiles for the circadian-controlled gene, Npas2, which has a peak expression at ZT22. Repair of the rhythmic Npas2 gene exhibits high amplitude transcribed strand (TS) (−s) repair peaks at 2 h, ∼24 h, and ∼48 h after drug injection. Only after 48 h does the nontranscribed strand (NTS) become the main source of repair product from the Npas2 gene. RPKM reads per kilobase per million reads. Adapted with permission from Yang et al. (104).

Figure 10

Genome-wide analysis of TS and NTS repair in host liver and human colorectal cancer xenografts.A, plots of average TS and NTS repair across all genes in mouse liver and in cisplatin sensitive (057) or resistant (119) xenografts. XR-seq data obtained at ZT0 are plotted as RPKM average repair reads (y axis) along the length of a “unit gene” (x axis). The unit gene was constructed using all nonoverlapping human or mouse genes >5 kpb with a distance >5 kbp between adjacent genes. The unit gene is 100 bins in length, and values for average repair were obtained by dividing each gene into 100 bins and averaging the repair values for each successive bin for all genes from 1 to 100. Average repair 2 kbp upstream and downstream was similarly obtained. B, Heatmaps (above) and radial diagram representations (below) of circadian TS and NTS repair cycles in host liver and in cisplatin sensitive (057) and resistant (119) xenografts. In the heatmaps, each horizontal line (1368 lines for liver) represents repair of one gene from ZT0 to ZT20 at six time points. Exp/Med is, for each gene, RPKM at a given ZT time point divided by the median ZT RPKM value. The criteria for selecting the significant cyclical genes both in TS and NTS is meta2d_pvalue<0.05, meta2d_rAMP>0.1. Based on this scale, 1368, 85, and 124 genes were cyclical in host livers, xenograft 057, and xenograft 119, respectively. In an additional cisplatin resistant xenograft, 413 (not shown), 48 genes were cyclical. The host liver radial diagram (left) indicates two peaks of repair in the TS, predawn and predusk, and the NTS radial diagram to the right exhibits a single peak at ZT8-10 corresponding to our previous data (see Fig. 7). In xenografts, the TS and NTS repairs are less coherent but tend to exhibit a single peak at ZT8-11, likely due to the peak of global repair activity as described in Figure 8. C, repair of two representative circadian-controlled genes, Dbp and Npas2. The screenshots illustrate repair in liver (left) and in the cisplatin sensitive xenograft 057 (middle). It can be seen that in the liver, repair of the circadian-controlled genes (Dbp, Npas2) follows their transcriptional oscillation, while the respective neighboring genes, Sphk, Rpl31, and Tbc1d8, show constant repair over the entire circadian cycle. The graphs to the right illustrate quantitative values for TS repair as a function of circadian time for the liver, and for one sensitive (057) and two resistant (119, 413) xenografts. In contrast to the liver, rhythmic repair of the circadian-controlled genes is absent in the xenografts. From Sancar (105).