Cross-talk between the circadian clock and the cell cycle in cancer

Abstract

The circadian clock is an endogenous timekeeper system that controls the daily rhythms of a variety of physiological processes. Accumulating evidence indicates that genetic changes or unhealthy lifestyle can lead to a disruption of circadian homeostasis, which is a risk factor for severe dysfunctions and pathologies including cancer. Cell cycle, proliferation, and cell death are closely intertwined with the circadian clock, and thus disruption of circadian rhythms appears to be linked to cancer development and progression. At the molecular level, the cell cycle machinery and the circadian clocks are controlled by similar mechanisms, including feedback loops of genes and protein products that display periodic activation and repression. Here, we review the circadian rhythmicity of genes associated with the cell cycle, proliferation, and apoptosis, and we highlight the potential connection between these processes, the circadian clock, and neoplastic transformations. Understanding these interconnections might have potential implications for the prevention and therapy of malignant diseases.

Key words::

• Apoptosis
• cancer
• cell cycle
• circadian clock
• MAPK cascade
• proliferation
• Wnt signaling

Key messages

• Epidemiologic studies have revealed a clear link between the disruption of circadian rhythms and cancer development in humans.

• Cell cycle, proliferation, and cell death are closely intertwined with the circadian clock.

• Many genes of cell cycle, apoptosis, and their regulatory networks, which are dysregulated during tumorigenesis, are rhythmically expressed during a 24-hour period.

Introduction

Organisms have to face daily repeating changes that occur in their surroundings at approximately the same time interval each day. To anticipate these changes and to co-ordinate the appropriate physiological and behavioral responses, many of these functions display daily rhythms. These rhythms are driven by an endogenous timekeeping system that regulates numerous biological processes in such a way that they repeat with approximately 24-hour periodicity and are thus called circadian rhythms. In mammals, the system is hierarchically organized and is composed of the central clock in the brain, which controls numerous clocks in the bodily cells of multiple tissues (neuronal and non-neuronal) and organs. The major role of the central clock is to ensure the synchrony of the endogenously generated rhythms entraining it to match the external environmental cycle, as well as to ensure their mutual synchrony within the entire organism. The most powerful signal that provides synchrony with daytime is light, even though other signals, such as temperature and diet, can reset the clock under specific conditions (1). To fulfill its role, the central clock is strategically positioned in the anterior hypothalamus, just above the optic chiasm, in the suprachiasmatic nuclei (SCN). Under natural conditions, the circadian clock properly couples the physiological and behavioral processes in the body with the external environment. However, changes in lifestyle that are associated with industrialization (light at night) and employment on an alternative work schedule (evening, night, rotation shift work) may disrupt the endogenous rhythmicity. Epidemiological studies have revealed a link between shift work and some cardiovascular, metabolic, gastrointestinal, and mental disorders (2) as well as some types of cancer (3–8). Similarly, experimental disruption of circadian homeostasis in experimental animals has also been associated with increased tumorigenesis and the acceleration of tumor growth (9–13). On the basis of human epidemiological studies and sufficient experimental evidence in animal models, the International Agency for Research on Cancer categorized ‘shift work that involves circadian disruption’ as most likely carcinogenic to humans (14). Tumor progression is mediated by the activation of transcriptional programs that are responsible for decreased apoptosis and increased cancer cell proliferation; these programs can be characterized as an unscheduled process determined by the activation of the cell cycle machinery in the presence or absence of mitogenic or anti-mitogenic stimuli, respectively. At the molecular level, the cell cycle machinery and the circadian clocks are controlled by similar mechanisms, such as gene feedback loops and their protein products that display periodic activation and repression. Therefore, the purpose of this review is to highlight the interactions between the circadian system and the regulation of the cell cycle machinery during tumorigenesis in mammals. Moreover, this review focuses on providing meta-analysis of published results obtained from microarray studies performed in various tissues to search for the most rhythmically expressed genes encoding regulators of the cell cycle, proliferation, and apoptosis.

Molecular organization of the cell cycle

The basic regulation of the cell cycle in mammals is a very complicated process that depends on dynamic changes in gene expression that are controlled by a regulatory network composed of the cyclin-dependent kinases (CDK), cyclins (Cyc), and CDK inhibitors. Cell cycle progression is governed by the action of the CDKs and the accumulation of their regulatory subunits, the Cycs, which are essential for proper kinase function. The subsequent varying levels of the particular Cycs (A, D, E, etc.) and their binding to the corresponding CDKs lead to the activation of the CDK/Cyc complexes that are further regulated by phosphorylation and protein interaction events that tightly control the timing and extent of CDK activation. It is followed by the initiation and progression of particular cellular responses, finally directing cells to divide. While most Cycs promote CDK activities, CDK inhibitors restrain their activity and halt cell cycle progression under unfavorable conditions.

The initiation of cell cycle progression is controlled by extracellular mitogenic signals that activate early genes, such as c-Myc and E2f, via various signaling cascades involving the MAPK (mitogen-activated protein kinases) and β-catenin pathways in a cell type-specific manner (15). According to the ‘classical’ model of the mammalian cell cycle, mitogenic stimuli drive the cell cycle through G1 phase through the expression of CycD, which binds to CDK4 and CDK6. Activation of these kinases leads to E2F-dependent transcription of a large set of components that support DNA replication and subsequent processes. E2F transcription factors operate either as transcriptional activators or repressors and are inhibited by the pocket proteins of the RB family (16). An increase in the amount of various types of CycD by mitogenic stimuli activates the CDK4/6 which leads to the phosphorylation of the RB family proteins, leading to their release from the E2F complexes. This leads to the activation of E2F proteins and the expression of a cluster of genes encoding cell cycle regulators including CycE and CycA. The G1/S transition is initiated by the accumulation of CycE, which binds to CDK2. The CDK2/CycE complex provides a positive feedback loop, which results in cell cycle progression. This basic concept of a cell cycle driven by specific CDKs was derived mainly from experiments using cultured cells, and in vivo experiments using knockout mice revealed a more complicated picture (17).

Upon G1/S transcriptional activation, the cells progress into the S phase, initiate DNA replication, and decrease transcription via negative feedback loops. The G1 phase CDK2/CycE complex and S phase CDK2/CycA complex target the CDK inhibitor p27 for degradation, and the subsequent increase in CDK2 activity inactivates E2F-mediated transcription of various genes, including the genes encoding CycE and CycA (18). The inhibitor p27 is an example of CDK inhibitors that interfere with the activities of various cyclin-dependent kinases, mediate cytostatic signals, and induce cell cycle arrest (15,19). The inhibitors belong to two classes: the Ink4 family (p15, p16, p18, p19) and the Cip1/Kip family (p21, p27, p57).

The accumulation of CycA during S phase appears to activate CDK2 which is no longer associated with CycE, and the CDK2/CycA complex phosphorylates numerous proteins that are considered to be required for the proper completion and exit from S phase (19). During the subsequent G2 phase, which is devoted to mending replication errors and ensuring that all is in order prior to mitosis, the A-type Cycs undergo proteolysis, and the B-type Cycs are actively synthesized and bind to CDK1. The CDK1/CycB complex is one of the master regulators of the centrosome cycle and triggers mitosis (17).

This complex is regulated by WEE1, a kinase that inactivates the CDK1/CycB complex, and thus regulates the transition from interphase to mitosis. During interphase, when CycB synthesis is activated, the CDK1/CycB complexes are kept inactive by the phosphorylation of CDK1 by WEE1. At the G2/M transition and mitotic onset, a sharp reversal of this phosphorylation is triggered by the CDC25 phosphatase, which acts as a major rate-limiting step in the activation of CDK1. Both WEE1 kinase and CDC25 phosphatase are tightly regulated to provide an accurate control of the mitotic onset. A variety of signaling pathways converge upon WEE1 and CDC25 to govern the G2/M transition including destabilization and degradation of WEE1 during G2 and M phases. All of these changes lead to the activation of the CDK1/CycB complex and entry into mitosis (20). Exit from mitosis requires the inactivation of the CDK1/CycB complex due to the degradation of the B-type Cycs via ubiquitination (17).

Proper replication of the genome and the prevention of translocation of damaged DNA into the daughter cells are ensured by specific transcription programs called the DNA damage checkpoints, which slow down or arrest cell cycle progression (21). Briefly, damage is detected by the ATR and ATM sensor kinases, with the aid of accessory proteins, and the signal is transmitted to the checkpoint kinases CHK1 and CHK2, respectively, which, together with ATM and ATR, reduce CDK activity by various mechanisms; some of these mechanisms are mediated by the activation of the p53 transcription factor or the downregulation of CDK activators such as the CDC25 phosphatases. Activation of the checkpoint pathways slows down or arrests cell cycle progression at the G1/S, intra-S and G2/M cell cycle checkpoints. An active ATM(ATR)/CHK2(CHK1) pathway leads to the stabilization of p53, a transcription factor whose key transcriptional target is p21. This pocket protein is a CDK inhibitor that blocks the CDK2/CycE complex and, thus, causes a G1 arrest. In contrast, the CDC25 phosphatase removes the inhibitory phosphorylations from the CDKs, and, therefore, downregulation of CDC25 leads to the inhibition of the CDK2/CycE(A) complex, which delays the G1/S transition. Similar to the G1/S transition, a checkpoint network operates the G2/M transition, whose critical target is the mitosis-promoting complex CDK1/CycB. The checkpoint pathways not only inhibit the activation of CDC25 but also facilitate the suppressive effect of WEE1 (20).

Mechanism of the circadian rhythmicity

In mammals, the circadian system represents an integral regulatory system composed of hierarchically organized circadian clocks. The regulatory complexity results from the fact that circadian clocks reside in nearly every (if not all) mammalian cell (22). These clock cells are equipped with a set of genes that are indispensable for circadian function, which are also called the clock genes. In mammals, Per1, Per2, Cry1, Cry2, Bmal1, Clock, Rev-erbα, Rora, and Csnk1d/e (encoding casein kinases 1δ/ε) have been recognized as clock genes (23). Peripheral clocks are regularly entrained by signals from the central pacemaker located in the SCN, which provides the other clocks with information about daytime and co-ordinates them accordingly. The clocks are also sensitive to various signals independent of the central clock that are derived from their local environment, such as metabolic signals. Situations in which the SCN and the locally derived signals are in conflict may lead to the malfunction of the clock and, consequently, to an impairment of temporal control of cell-specific programs.

The principle of the mechanism of how the circadian rhythmicity is generated seems similar for both the central and peripheral clocks. The basic components of the clockwork are those mentioned above, the clock genes that encode clock proteins. Absence or malfunction of any of the clock components leads to significant abnormalities in circadian rhythmicity (24,25). A contemporary model of the molecular core clockwork presumes that the rhythmical signal in the cell arises from the daily rhythmical presence and absence of the clock proteins, which function as transcriptional factors that rhythmically drive the expression of other, so-called clock-controlled, genes. Importantly, the rhythmical presence of the clock proteins is driven in a cell-autonomic fashion (26,27) via a complex mechanism, which has been only partially clarified. This mechanism is based on loops in which the rhythmically available clock proteins feed back onto promoters of the clock genes and activate or inhibit their expression; this, in turn, drives the rhythms in the protein levels. The clock proteins CLOCK and BMAL1 serve as transcriptional activators that switch on the transcription of genes that contain E-box response elements (CACGTG) in their promoters. E-boxes are present in the promoters of the clock genes Per1,2 and Cry1,2 and two orphan nuclear receptors Rev-erbα and Rora, as well as in the promoters of the clock-controlled genes, such as the genes that are not part of the core clockwork, but are controlled by it and transmit the rhythmical signal outside of the clock (see below). After switching on transcription of the Per1,2 and Cry1,2 clock genes, the PER1,2 and CRY1,2 proteins are formed with a clock protein-specific delay, accumulate in the cytoplasm, and form homo- and heterodimers. The dynamics of this checkpoint are controlled by post-translational modifications of the clock proteins, mainly by phosphorylation and subsequent proteasomal degradation. PER protein phosphorylation by casein kinase 1ε (CK1ε) and CK1δ facilities their ubiquitination and degradation. CRY protein is targeted for degradation by a member of the F-box family of ubiquitin E3 ligases, FBXL3 (28,29). Post-translational modifications contribute to the delay in the protein accumulation in the cytoplasm. Finally, the PER:CRY heterodimer enters into the nucleus (30,31) and inhibits CLOCK:BMAL1-mediated transcription, most likely through mechanisms involving directed histone deacetylation and other chromatin modifications (32). Later during the day, the PER:CRY heterodimer is degraded, and the repression is relieved, thus initiating the transcriptional activation of CLOCK:BMAL1 and a new transcriptional-translational cycle. The rhythmical transcription of CLOCK:BMAL1 is controlled via circadian oscillations in the transcription of Bmal1, which is driven by the binding of REV-ERBα and RORA to ROR-response elements (RORE) in the Bmal1 promoter. REV-ERBα and RORA repress and activate Bmal1 transcription, respectively (33,34). These interlocked positive and negative transcriptional-translational feedback loops repeat with a circadian period.

Apart from this canonical mechanism, other mechanisms may contribute to the generation of the circadian rhythm, namely those involving miRNAs (35). The miRNAs act as potent silencers of gene expression through translational repression of mRNA degradation. It has been shown that miR-219 is a target of the CLOCK:BMAL1 complex and exhibits robust circadian expression rhythms as a clock-regulated gene. Translation control via miRNAs may, therefore, represent a novel regulatory level of the circadian clock. Another regulatory mechanism might be related to the bZIP transcription factors, namely E4BP4, which is a key negative component of the circadian clock (36).

In summary, the basic regulatory mechanisms of the circadian clock appear to be similar to those of the ‘cell cycle clock’, but they differ in some fundamental aspects. The cell division rate is not strictly temporally controlled to operate on a 24-hour basis, and somatic cells can undergo long-term cell cycle arrest, which results in relative independence of the cell cycle from the circadian clock. However, substantial amounts of evidence have accumulated that document the mutual coupling of the cell cycle and the circadian clock, mainly via temporal control of the cell cycle regulators as putative clock-controlled genes.

Circadian expression of cell cycle genes

In general, the circadian clock generates the rhythmic signal via the above-described mechanism, and the signal is passed on to the cellular processes by at least two mechanisms. The first mechanism is mediated via the E-boxes in the promoters of genes involved in cellular regulatory pathways. The rhythmic presence of the clock transcriptional activator, the CLOCK:BMAL1 complex, which binds to this sequence, may rhythmically switch the transcription of these genes on and off (27). Because the CLOCK:BMAL1 heterodimer is transcriptionally active during the first part of the circadian cycle (i.e. during the subjective day), the genes temporally regulated via this pathway exhibit high expression during the day and low expression during the night. The other pathway is mediated via the RORE sequence in the promoters of some genes. As described above, the RORE is a target of the rhythmically appearing clock protein REV-ERBα, which inhibits the transcription of the genes. Because REV-ERBα levels are high during the day and low during the night, genes that are temporally regulated by this mechanism are expressed in a manner opposite to those regulated by the E-boxes (33). These two basic mechanisms allow for the rhythmic transcription of sets of genes in a phase-specific manner.

Numerous gene expression microarray studies have indicated ample evidence that circadian rhythmic genes are associated with cell cycle progression, proliferation, and apoptosis. We identified all of the genes with these activities according to the Gene Ontology consortium system (37) and then examined the circadian rhythmicity of these genes using publicly available microarray data (38,39). From 39 microarray data sets, 19 distinct tissues were selected, and the tissue was considered rhythmic if at least one significant rhythm of a particular gene was observed. The results are summarized in Supplementary Tables I–IV (to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296), and a condensed version can be found in Figures 1–4. These findings indicate that some of these genes are rhythmically expressed in more than one tissue. Most prominent were the genes Cdkn1a, Cdkn1c, and Cdkn1b, encoding the CDK inhibitors of the Cip/Kip family (p21, p57, p27), which were shown to be rhythmic in several tissues (Supplementary Table I to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296, Figure 1). The circadian rhythmicity of a group of cyclins is of particular interest due to their fluctuation during the cell cycle. Among the cyclins whose functions in the cell cycle are well established (see above), the genes encoding CycD1 (Ccnd1), CycD2 (Ccnd2), CycA1 (Ccna1), CycA2 (Ccna2), and CycE1 (Ccne1) exhibit rhythmicity in several tissues (Supplementary Table I to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296, Figure 1). Similarly, the rhythmic expression of CycE, CycA, and CycB1 was observed in human oral mucosa, with CycA and CycB1 levels peaking 1 h and 6 h later than CycE, respectively (40), and their expression patterns were synchronized with the circadian oscillations of clock gene expression (41). Remarkably, the expression of the non-canonical cyclins, whose roles and relationships to cell cycle regulation are poorly understood, also exhibited circadian rhythm in a marked number of tissues (Supplementary Table I to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296, Figure 1). The expression of genes encoding regulatory proteins, in particular WEE1 kinase, GADD45α, GADD45β, and G0S2, was also rhythmic in many tissues (Figure 1); these proteins are hot candidates for linking the cellular clock to cell cycle. As shown in Supplementary Tables II–IV to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296 and Figures 2–4, some elements of regulatory pathways, in particular those involving the Wnt/β-catenin, MAPK signaling, and apoptotic pathways, showed rhythmicity and, thus, might also play roles in the communication between the circadian clock on one side and cell cycle, proliferation, and apoptosis on the other side.

Figure 1. Frequency of representative rhythmically expressed genes in the functional category Cell cycle selected according to the Gene Ontology classification [GO:0007049]. The rhythmicity was compared in whole brain, prefrontal cortex, suprachiasmatic nuclei, brain stem, cerebellum, hypophysis, adrenal gland, aorta, heart, skeletal muscle, liver, kidney, brown adipose tissue, white adipose tissue, bone, lung, colon, and 3T3 cell line. The detailed data of all genes of the Cell cycle cluster and the incidence of their rhythmicity in the particular tissues are summarized in Supplementary Table I (to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296).

Figure 2. Frequency of representative rhythmically expressed genes in the functional category Wnt receptor signaling pathway selected according to the Gene Ontology classification [GO:0016055]. For further details, see Figure 1, and also Supplementary Table II (to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296).

Figure 3. Frequency of representative rhythmically expressed genes in the functional category MAPK cascade selected according to the Gene Ontology classification [GO:0000165]. For further details, see Figure 1, and also Supplementary Table III (to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296).

Figure 4. Frequency of representative rhythmically expressed genes in the functional category Apoptotic processes selected according to the Gene Ontology classification [GO:0006915]. For further details, see Figure 1, and also Supplementary Table IV(to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296).

In summary, the data summarized in Supplementary Tables I–IV to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296 and Figures 1–4 show that some of the genes participating in the regulation of cell cycle, proliferation, and apoptosis might be potential targets of the circadian clock. However, the circadian rhythmic expression of these genes was identified predominantly using microarray analyses, and only some of these data were validated using the more sensitive real-time PCR assay. Moreover, direct transcriptional control by the circadian clock proteins has been proven only for a minority of these genes thus far. Although the definitive experimental evidence that these genes are direct transcriptional targets of the circadian clock is still mostly lacking, the data summarized in this review support their roles in the communication between the circadian clock, the cell cycle, proliferation, and apoptosis. Moreover, these data suggest that their deregulation may play a causal role in tumorigenesis.

As mentioned above, the presence of E-boxes in a gene promoter represents a fundamental feature of the circadian control of gene expression, and, therefore, evidence of this sequence in the promoter region indicates that such genes might be under circadian regulation. However, the evidence of E-box presence per se is not definitive evidence for the circadian regulation of gene transcription. Promoters of cell cycle regulatory elements, in particular the cyclins, can respond primarily to signals derived from the cell cycle, but they might also be affected by clock-related signals. Thus, these genes respond to both intrinsic regulatory mechanisms, as well as to signals from the local environment, through the selective utilization of an E-box as a regulatory element of circadian control. Such regulation was observed in the case of the gene Ccnb1 gene encoding CycB1, which has an E-box-containing promoter, but its E-boxes do not appear to be responsive to BMAL1:CLOCK (42), in spite of the fact that CycB1 levels oscillate.

Cell proliferation is temporally controlled

For a long time, it has been known that cell proliferation in adult, rapidly renewing tissue is a temporally regulated process. Circadian variations have been detected for thymidine uptake, mitotic figures, and DNA content (43–46). Intriguingly, the rhythms in proliferation are phase-locked relative to daytime. The S (DNA synthesis) and M (mitosis) phases of the cell cycle usually coincide with the active period of the day; for example, in nocturnal laboratory animals, the S and M phases occur during the night period (44). In contrast, in the human epidermis and rectal and oral mucosa, DNA synthesis peaks in the light phase of the day (43,45,47) and is, thus, in the opposite phase compared to the rhythms in mice and rats. Additionally, the peak of the S phase, as determined by analysis of the cyclins, is reached in human oral mucosa cells in the middle of the active, light period of the day (i.e. during the daytime) (40). In accordance with the diurnal rhythmicity of proliferation, tissues also exhibit circadian variations in their susceptibilities to extracellular mitogenic signals, such as growth factors (48). These findings indicate that cell proliferation depends not only on extracellular mitogenic signals, but is also temporally controlled by the circadian clock, and interaction of these factors may have both physiological and pathological implications.

Proliferation of tumor cells often follow a cyclic pattern that differs from normal tissue (49–51), and the disruption of the clock functions modulate rhythmic mitosis, cell proliferation, and tumor growth (52–56). For example, the mouse epidermis is more susceptible to UVB radiation during the night when its cells are more proliferative; consequently, UVB radiation induces the development of more squamous tumors when administered during the night than during the day (57).

The comparison of the mouse transcriptome of wild-type mice and animals with malfunctioning circadian clocks due to mutation in the Clock gene showed that up to 16% of all rhythmic genes are associated with the cell cycle and that Clock mutation upregulates genes that encode cell cycle inhibitors, and downregulates pro-proliferative genes (58). Similarly, disruption of the circadian clock via mutations in Bmal reduced proliferation in murine epidermis, which is in accordance with the simultaneous role of CLOCK and BMAL1 as positive elements of the molecular clockwork (see above).

In contrast, mutation of the negative elements of the feedback mechanism, such as the Per1/2 genes, increased proliferation (59). Additionally, mutations in some components of the circadian clock were found to reduce the efficiency of liver regeneration (60), display a tumor-prone phenotype (61), accelerate tumorigenesis (55), or increase cancer risk (62). These findings indicate that circadian regulation plays a role in the control of proliferative signals, as well as in tumorigenesis.

Interplay between the cell cycle, circadian clock, and tumorigenesis

Regulation of G1/S transition

The circadian control of the Ccnd1 gene encoding CycD1, which controls G1 phase in complex with CDK4 and CDK6, was demonstrated in the majority of the investigated tissues (Figure 1), and its overexpression in breast cancer positively correlated with poor prognosis (63). This indicates functional importance of the circadian rhythmicity of CycD1 and its role in tumorigenesis. Mutations in Per1 and Per2 cause significant deregulation of Ccnd1 expression, which leads to a shortening of the cell cycle and to the elevation of the proliferation rate (61,64). Additionally, in Per2 mutant mice, radiation induces the development of lymphomas at a higher rate than in the wild-type animals (61). Similarly, the ApcMin/+ mice (colorectal neoplasia-susceptible mice) crossed with Per2 mutant mice have a higher incidence of intestinal polyps than the ApcMin/+ mice alone (55). The upregulation of CycD1 is indirectly promoted by the inhibition of c-Myc transcription (61), an oncogene that plays a key role in cellular proliferation and tumorigenesis (65). The promoter of this gene contains an E-box that can be bound by different transcription factors including CLOCK and BMAL1 (65). It was shown that a close homolog of CLOCK, NPAS2, which forms a complex with BMAL1, can suppress the activity of the c-Myc promoter (61). Similarly, epigenetic inactivation of Bmal1 impairs the circadian expression pattern of c-Myc in association with a loss of BMAL1 occupancy at their respective promoters (53). Additionally, overexpression of Per1 results in elevated levels of c-MYC in colon cancer cell lines after irradiation (66), and the mutation S662G in Per2 enhances tumorigenesis in mice with a cancer-sensitive background (62). However, the antiproliferative and tumor- suppressing effects of the PER transcription factors are not likely to be associated with c-MYC in all cell types. For example, in mammary cancer cells, the downregulation of Per1 gene expression increases the expression of genes encoding CycD and CycE and increases cell proliferation (67), which indicates that there might be differences in the interactions between the circadian clock and the cell cycle in various cell types. Importantly, c-Myc and Per1 transcript levels follow the same rhythmic pattern (68), although the CLOCK:BMAL1 heterodimer regulates Per1/2 and c-Myc expression in an opposite manner; CLOCK:BMAL1 induces the transcription of the Per genes but inhibits the transcription of c-Myc (61). In addition to c-Myc, another gene encoding a MYC protein family, N-Myc, was shown to be controlled by the clock proteins REV-ERBα and RORA (69).

Circadian regulators might target through regulation of c-Myc not only Ccnd1, but also the genes encoding the transcription factors (Gadd45aα, Cdk4, Trp53, Mdm2; see below), and the CDK inhibitors p21, p27, and p57 (Cdkn1a, Cdkn1b, and Cdkn1c) (70–72). These inhibitors prevent premature entry into S phase, tie the G1/S transition to regulatory inputs (15), and are often deregulated in human cancers (73). The screening of the microarray data unequivocally showed that one of the highest incidences of a circadian pattern among all of the studied cell cycle-related genes belonged to Cdkn1a, followed by Cdkn1c and Cdkn1b (Figure 1). The circadian regulation of the Cdkn1a gene is consistent with the finding that the Cdkn1a is upregulated and the proliferation state is changed in Bmal1-deficient mice (74,75). Detailed analysis of Cdkn1a expression in hepatocytes showed that the circadian expression of this gene is indirectly regulated by BMAL1 through the Roraα/Rev-erbaα pathway and that p21 negatively regulates G1 phase progression (74). Furthermore, BMAL1 depletion in a tumorigenic mesothelial cell line resulted in the disruption of the cell cycle in association with the downregulation of p21 and the upregulation of CycE expression (76).

C-MYC is able to regulate the expression of genes encoding CDK inhibitors from the Cip/Kip family (p21, p27, p57), as well as the gene encoding CDK inhibitors from the INK4 family (71,72). As summarized in Figure 1, the members of this family also exhibit circadian rhythmicity in some tissues [Cdkn2b(p15) > Cdkn2c(p18) > Cdkn2a(p16) = Cdkn2d(p19)] but with a lower incidence than the Cip/Kip family. The INK4 inhibitors, which primarily target CDK4 and CDK6, are often deregulated in cancer (17). The meta-analysis also shows that further regulators of G1/S transition, such as cyclins (CycD1, and much less CycE), and the CDKs themselves exhibit circadian regulation in several tissues. As shown in Figure 1, the genes encoding CDK2 (Cdk2), CDK4 (Cdk4), and CDK6 (Cdk6), which control the G1/S phase of the cell cycle, exhibit circadian rhythmicity. Similarly, circadian rhythmicity was also found in the case of the CDC25 phosphatases encoded by the Cdc25a and Cdc25b genes (Figure 1), which remove the inhibitory phosphorylation on the CDKs and, thus, promote G1/S and G2/M progression; the overexpression of these phosphatases has been frequently found in cancer cell lines and in cancer samples from patients with poor prognosis (77).

These data indicate that many genes operating in the G1/S phase of cell cycle exhibit circadian rhythmicity and that c-Myc might be one mechanism by which the circadian clock controls tumorigenesis. The link between the circadian clock and the cell cycle machinery is ensured primarily by Bmal1, even if some studies indicate that the Bmal1 and Clock genes are associated with the regulation of the cell cycle. Mutation in the Clock gene resulted in the upregulation of Cdkn1a and Cdkn1b and the downregulation of Ccnd3, Ccne1, and Cdk2 genes, which encode p21, p27, CycD3, CycE1, and CDK2, respectively (58).

DNA damage checkpoints

Different cellular stress responses initiated by DNA damage, hypoxia, and hyperproliferative oncogenic signals are integrated at the level of the p53 protein, encoded by the Trp53 gene; this gene exhibits circadian rhythmicity in various tissues (Figure 1), and its product, the p53 protein, was observed to be rhythmic in human oral mucosa (40). In its role as a transcription factor, it activates a series of genes that can restrict cell cycle progression and trigger DNA repair and apoptosis. Among the known p53 transcriptional targets is Mdm2, which encodes E3 ubiquitin ligase MDM2, an enzyme that acts in a feedback loop to antagonize p53 function and exhibits circadian expression in mouse liver (61). The role of BMAL1 in the regulation of the p53 pathway has been recently proven. Downregulation of Bmal1 expression attenuates the p53-dependent induction of Cdkn1a and releases the cells from the cell cycle arrest (78). Additionally, the genes Trp53, Mdm2, and Gadd45a are genes responsive to the MYC transcription factors (70,71), which indicate the possible role of BMAL1 in their circadian regulation. Furthermore, the deregulation of Gadd45aα and Mdm2 rhythmicity in Per2-deficient mice (61) supports the role of the circadian clock in the control of the p53 pathway.

A direct link between the above-mentioned genes or their protein products and the tumorigenesis has been repeatedly demonstrated. The p53 protein upregulates Gadd45aα, whose transcript levels are increased following stressful growth arrest conditions, and interacts with PCNA, a protein of the DNA polymerase enzyme complex, p21, and the CDK1/CycB complex. Emerging functional evidence implies that GADD45 proteins serve as tumor suppressors in response to diverse stimuli and potentially act through the p53-mediated apoptotic pathways, which are inactivated in the majority of cancers (79). Similarly, Dmtf1, which encodes the tumor suppressor cyclin D-binding myb-like transcription factor 1, exhibits circadian rhythmicity in many tissues (Figure 1). DMTF1 directly binds to the Arf promoter to activate the expression of CDK inhibitors that inhibit the activity of the CycD-dependent kinases or stabilize and activate the MDM2-p53-dependent cell cycle arrest. Tumors from Dmtf1-deficient mice show a significant downregulation of Arf and Cdkn1a and display p53 inactivity and more aggressive phenotypes than tumors without Dmtf1 deletion (80).

While the expression of key positive cell cycle regulators, including c-Myc, are increased together with proliferation in the Per1 and Per2 mutant mice, the overexpression of Per2 leads to cell cycle arrest (81). The most prominent pathway leading to G1 arrest and apoptosis is associated with p53-dependent signals that can be modulated by PER1 proteins through the ATM and CHK2 kinases (66). Additionally, PER2 downregulation delays the DNA damage-induced CHK2 activation and overrides DNA damage-induced apoptosis and cell cycle arrest (82). In contrast to the ATM/CHK2 pathway, the connection of the circadian clock to the ATR/CHK1 checkpoint pathway seems to be coupled to the accessory mammalian clock protein TIM, which has been shown to be tightly related with the molecular clock mechanism (83). Similarly, TIM is required for ATM-dependent activation of Chk2 and the G2/M checkpoint arrest, although TIM is not essential for ATM activation (84). It was recently shown that knockdown of TIM in mammalian cells shortens the clock period and that it interacts with both CHK1 and CRY1, thus connecting the circadian clock with the cell cycle (85). These findings indicate that the core clock proteins can interact with checkpoint proteins and modulate proliferation and/or tumorigenesis.

Regulation of G2-M transition

Similar to the G1/S transition, the G2/M transition is also controlled by the PER-mediated ATM/CHK2 pathway (66); however, the main linkers between the circadian clock and cell cycle control during G2/M phase seem to be WEE1, a tyrosine kinase that inactivates the CDK1/CycB complex, and CDC25 phosphatase, which removes the inhibitory phosphorylation of CDK1, thus promoting G2/M progression. As summarized in Figure 1, circadian rhythmicity of Wee1 and Cdc25a was identified in a majority of the investigated tissues (Supplementary Table I to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296).

The gene encoding WEE1 contains three E-boxes that bind BMAL1:CLOCK heterodimers to increase transcriptional activity, whereas the CRY:PER heterodimers have the opposite effect. Using a model of liver regeneration, Matsuo and colleagues (60) provided evidence that supports the circadian regulation of the cell cycle via this pathway. They demonstrated that 1) the circadian clock gates the entry of the cells into the G2/M phase, 2) cell division is impaired in Cry1/2 double-knockout mice, and 3) the timing of the G2/M transition depends on the clock-controlled Wee1. Additionally, Bmal1 knockdown in malignant pleural mesothelioma cells decreased the levels of WEE1 and CycB (76).

The oncogenic role of WEE1 is supported by the downregulation of Wee1 mRNA in patients with malignant melanoma (86) and the significant changes in Wee1 expression during tumorigenesis (67,87,88). The original robust intestinal circadian oscillations of Wee1 expression in colon tissue (89,90) was significantly decreased during colonic tumorigenesis (88). Therefore, the downregulation of WEE1 might lead to the release of the cell cycle arrest during the G2/M transition.

However, a role for some transcription factors regulated by circadian clock also has to be considered. Previous studies have shown that CDC25A is a physiologically relevant transcriptional target of c-MYC, and GADD45 proteins dissociate the CDK1/CycB1 complexes, thus damping the G2-M transition (91). The genes encoding these proteins exhibit circadian rhythmicity in several tissues (Figure 1).

Non-canonical cyclins

The data summarized in Figure 1 indicate that, with the exception of CycD1, the transcripts of the other canonical cyclins of cell, namely CycA, CycB, and CycE, show less frequent putative diurnal rhythmicity than the unconventional cyclins (Figure 1), which regulate distinct processes, such as RNA transcription and apoptosis. Together with Ccnd1 encoding CycD1, the meta-analyses indicated the highest incidence of the circadian rhythmicity in the transcription of the Ccnl2 gene, which encodes CycL2, a member of the cyclin family that has been implicated in the regulation of cell cycle progression and/or transcriptional regulation; CycL2 has been shown to have anti-proliferative effects due to G0/G1 arrest and apoptosis (92). Lower incidence of circadian rhythmicity was found in the cases of the genes encoding CycL1 (Ccnl1), CycC (Ccnc), and CycT1 (Ccnt1) and for the genes encoding their respective CDKs, Cdk11, Cdk8, and Cdk9 (Figure 1). All of these genes encode proteins that have been predominantly linked to the regulation of RNA polymerase II during initiation, elongation, and RNA processing. These cyclins and their kinases control transcription in a manner analogous to the regulation of the cell cycle, and the putative rhythmicity of the CDK/cyclin complexes is in agreement with the widespread diurnal rhythms of mRNA expression. It is well known that CycC and CDK8 are frequently deregulated in various human cancers, and CDK8 regulates β-catenin in colonic mucosa (93). Similarly, CycL1 has been identified as an oncogene in head and neck cancer that can regulate G0/G1 cell cycle progression following induction by several growth factors (94).

Relatively frequent circadian rhythmicity was also found in genes encoding several other cyclins (Figure 1). CycI protein, which is encoded by the Ccni gene, has been recently shown to oscillate during the cell cycle, and its depletion by siRNA led to the accumulation of cells in G2/M phase (95). Apart from its putative role in the cell cycle, CycI can also exert its influence on apoptosis via regulators of the Bcl family (96). As shown in Figure 4, the Bcl2 genes exhibit circadian rhythmicity in the majority of the investigated tissues (Supplementary Table IV to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296). The mammalian CycG1, CycG2, and CycF, which are encoded by the Ccng1, Ccng2, and Ccnf genes, are examples of unconventional cyclins, whose roles in the regulation of the cell cycle is not well understood. However, Ccng1 is overexpressed in several human cancers, suggesting it plays a positive role in cell cycle regulation (97), and expression of CycG2 and CycF correlate with gastric cancer progression (98) and the size of hepatocellular carcinomas (99), respectively.

Rhythmicity was found also in the Ccny gene encoding CycY (Figure 1), a cyclin that has been implicated in the control of Wnt/β-catenin regulatory pathway. CycY is a regulatory subunit of CDK14 that acts as a regulator of the Wnt signaling pathway, and this protein may link cell cycle regulators and Wnt signaling (100).

Wnt- and TGFβ-signaling, circadian clock, and cancer

The initiation of the cell cycle is strongly controlled by extracellular signals that induce intracellular signaling cascades whose disarrangement is frequently associated with tumorigenesis. These signaling systems (Wnt, Notch, Hedgehog, TGFβ) secrete signaling molecules from one cell or tissue to activate the surface receptor tyrosine kinases in the neighboring cells and tissues and, thus, regulate various cell processes including proliferation and cell survival. The published microarray data indicate the possibility of partial circadian control of cell cycle progression via these pathways. For example, promoter analysis revealed that some of the genes in the Wnt, Notch, and TGFβ pathways contain putative BMAL:CLOCK-binding sites within their promoter (59,101). Bmal1 knockdown attenuates Wnt signaling activity and expression of Wnt-related genes, whereas Bmal1 overexpression augments this activity (59,101). Recently, it has been reported that Bmal1 knockdown is associated with lower levels of Wnt-related genes compared to the controls and that the expression of Wnt-related factors varies with a 12-h period in wild-type mice, but not Bmal1 knockdown animals (59).

To analyze the putative circadian regulation of the Wnt pathways, we used Gene Ontology system to categorize Wnt-associated genes and found that a large quantity of these genes exhibit circadian rhythmicity in various tissues (Supplementary Table II to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296). Based on these results, the principal components of the Wnt pathways and their positive and negative regulatory elements, which showed circadian rhythmicity in more than five tissues, are summarized in Figure 2. Interestingly, the deregulation of many of these components is considered to be linked with cancer initiation and progression in various tissues (102,103). The relationship between the circadian clock, the Wnt pathway, and tumorigenesis has been recently demonstrated in nude mouse xenograft models, where circadian disruption accelerated tumor growth through the Wnt pathway (104).

The canonical Wnt/β-catenin pathway consists of Wnt ligands that bind to the transmembrane receptor Frizzled (FZD) to activate receptor oligomerization with the Wnt co-receptor LRP5/6. This leads to the stabilization of intracellular β-catenin and its translocation into the nucleus, where it acts as a co-activator of transcription. The formation of the trimolecular complex of Wnt-Frizzled-LRP5/6 leads to the phosphorylation of LRP5/6 by protein kinases, the recruitment of the Dishevelled (DVL) protein, and the degradation of the protein Axin. These reactions lead to the inactivation of the destruction complex, which phosphorylates β-catenin and targets it for degradation by the proteasome in the absence of Wnt (100). FZDs and DVL are not only components of the canonical Wnt/β-catenin pathway, but they are also indispensable for the non-canonical Wnt/PCP (planar cell polarity) and Wnt/Ca²⁺ pathways, where WNT signals are transduced through FZD receptors and RYK and ROR co-receptors to the DVL-dependent (Rho family GTPases, MAP kinases) or Ca²⁺-dependent (Nemo-like kinase, NLK) pathways (102). The data summarized in Figure 2 show the circadian rhythmicity of genes encoding some WNT signals (Wnt4, Wnt5b, Wnt7b), FZD receptors (Fzd1, Fzd3, Fzd6, Fzd7), their co-receptors (LRP5, Ryk), catenins (Ctnnb1, Ctnnd1), and other core components of canonical and non-canonical pathways encoded by Celsr2, Axin2, Dvl1, Dvl2, Prickle1, and Vangl2 genes. Of note, the meta-analysis also indicated a high incidence of circadian rhythmicity in the positive and negative regulators of the Wnt pathways. In particular, rhythmicity was observed for genes that inhibit Wnt signaling upstream of β-catenin (Apcdd1, Dab2, Invs, Csnk1a1, Gsk3b, Lrp4, Wwox, Nlk, Cul3), including those that are secreted into the extracellular space, where they sequester Wnt molecules or form inactive complexes with the FZD receptors (Sfrp5, Frzb, Sfrp2). Regarding the positive regulators of the Wnt pathway, circadian rhythmicity was observed for kinases that phosphorylate the LRP6 co-receptor (Grk5, Csnk1d, Csnk1e, Csnk1a1), as well as for factors that promote this phosphorylation (Caprin2, Lgr6, Lgr4).

Several avenues of cross-talk between the Wnt pathway and the cell cycle machinery have been demonstrated. Activation of the Wnt pathway changes during the cell cycle and peaks at the G2/M transition (105), which indicates some relationship between these two processes. Consistent with the concept of a direct link between the cell cycle and Wnt signaling are findings that show amplification of β-catenin-driven gene transcription by the CDK8/CycC complex and the enhancement of Wnt signaling transduction via the Wnt receptor and co-receptor by the CDK14/CycY complex (100). Similarly, c-MYC and CycD1 have been reported as targets of the Wnt/β-catenin pathway (106,107). Finally, some casein kinases (CKs), which play complex roles in Wnt signaling, have been implicated in oncogenesis (108,109) and in the post-transcriptional modulation of the core clock element (see above). Activity of some casein kinases requires the p120-catenin (δ-catenin), which is encoded by the Ctnnd1 gene (110). This protein facilitates ubiquitin-mediated proteolysis of the CDC25A phosphatase throughout interphase and after exposure to genotoxic stress (111). As shown in Figures 1 and 2, the circadian rhythmicity of Ctnnd1 and Cdc25a was observed in various tissues.

Some of the genes on the microarray that belong to the Gene Ontology class ‘Wnt pathway’ and are differentially expressed during the 24-hour clock period have been proposed as avenues of cross-talk between the transforming growth factor-β (TGFβ) and Wnt pathways (Supplementary Table II to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296, Figure 2). This is obvious for the genes encoding TGFβ-responsive transcriptional modulator SMAD3 (Smad3), SMAD interacting protein 1 (Zeb2), and kinase MARK2 (Mark2). Additionally, the genes encoding TGFβ (Tgfb1, Tgfb3, Tgfb2) and its receptor (Tgfbr1) are also rhythmically expressed in several tissues (Figure 3). TGFβ signaling induces a G1 phase cell arrest via SMAD3, which promotes cell cycle arrest through its repression of c-MYC and induction of p15 and p21, thus inhibiting the CDK4,6/CycD and CDK2/CycE complexes (112). Further evidence for possible cross-talk between the TGFβ pathway and circadian clock comes from studies in which the promoter activities of Smad3 and other components of the TGFβ regulatory cascade were upregulated by CLOCK:BMAL1 (59,113,114) and the TGFβ receptors were downregulated by Clock mutation (58).

MAPK signaling cascades, cell proliferation, and circadian rhythmicity

Many extracellular signals that regulate proliferation, including growth factors and signals from non-canonical Wnt pathway (see above), are transduced from the cell surface to the nucleus via the MAPK signaling cascades. The terminal kinases are activated via three-tiered signaling modules that are composed of MAP kinase kinase kinases (MAP3Ks or MKKKs), MAP kinase kinases (MAP2Ks or MKKs), and MAP kinases (MAPKs or ERKs, JNKs, p38 proteins). Both the ERK1/2 and JNK/p38 pathways have been shown to play essential roles in cell cycle progression in response to various signals, including extracellular mitogenic stimuli and stressors, such as UV radiation, oxidative stress, etc. Additionally, many of these kinases appear to possess anti-oncogenic or pro-oncogenic properties in cell culture and animal models (115).

As shown in Supplementary Table III to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296 and Figure 3, several members of a wide group of these kinases exhibit circadian rhythmicity in their transcripts within numerous tissues. The highest incidence of rhythmicity was identified in genes encoding kinases in the JNK/p38 module, in particular Map3k6 (ASK2), Map2k7 (MKK7), Map2k3 (MKK3), and Map2k6 (MKK6), all of which are upstream activators of the JNK and p38 pathways, and in genes encoding their substrates, Mapk12 (p38γ), Mapk14 (p38α), and Mapk10 (JNK3). In contrast, the incidence of circadian rhythmicity in the kinases operating in the ERK1/2 module was lower, by more than 50%, and included Mapk1 and Map2k2, which encode ERK2 and MKK2, respectively. Many studies have shown the role of these kinases in cell cycle progression. Mouse embryonic fibroblasts lacking Map2k7 (Map2k7^(−/−)) exhibit defects in proliferation, premature senescence, and G2/M cell cycle arrest (116). Moreover, MKK7-mediated JNK activation causes the phosphorylation of PER2 and, thus, increases its half-life (117), whereas the downregulation of Map3k6 expression significantly suppresses tumor growth in vivo (118). Mapk12 contributes to the survival of UV-treated cells and is essential for the optimal activation of checkpoint signaling in response to UV (119); deletion of Mapk12 is accompanied by a prolonged S phase cell cycle arrest (119) and the induction of an M phase arrest (120). Moreover, a transient increase in the clock gene Per1 via the activation of tumor necrosis factor (TNF) receptor 1 is partially dependent on p38 MAPK signaling (121). A relatively high incidence of circadian rhythmicity was found also in Mapk6 which encodes the atypical MAPK, ERK3; this protein was recently shown to be involved in cell cycle regulation and tumorigenic processes (122).

As mentioned in the previous section, MAPKs are also indispensable for non-canonical Wnt signaling when the WNT signals are transduced through the DVL-dependent or Ca²⁺-dependent sub-pathways, which transduce the Wnt signals to the JNK cascade or to calcium/calmodulin-dependent kinase II (CaMKII), respectively (102). CaMKII induces the activation of the MAP kinases TAK1 and NLK, which are encoded by the genes Map3k7 and Nlk, respectively; both of these genes show circadian rhythmicity in their expression patterns in several tissues (Figures 2 and 3).

The first step in the activation of the MAPK cascades usually consists of relieving the inhibition of the MAP3Ks by smaller ligands, such as the proteins of the growth arrest and DNA damage-inducible 45 (GADD45) family, which are encoded by the Gadd45α, Gadd45β, and Gadd45γ genes. All three GADD45 family members directly interact with the kinases upstream of the JNK/p38 module in response to environmental stress, resulting in apoptosis or the stimulation of pro-survival events (79). Deregulation of these proteins and their genes has been observed in various types of cancer (79), and some studies have proposed a relationship between the GADD45 family and the circadian clock. The Cry-silenced cell line exhibited decreased levels of Gadd45α (123), and impaired circadian expression of Gadd45α was observed in the Per mutant mice (61). This is in agreement with our finding of a high incidence of Gadd45 circadian rhythmicity in 12 from 19 tissues (Supplementary Table I to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296, Figure 1).

Apoptosis

Tissue growth depends on the equilibrium between proliferation and cell death. Apoptosis, a form of programmed cell death, is one of the most important mechanisms determining cell fate. Sustaining homeostasis depends on the right balance between proapoptotic and antiapoptotic signals (124), and disruption of the apoptotic regulatory pathways is one of the important causes of cancer initiation (125). Insufficient apoptosis caused by defects in proper proapoptotic signaling or increased activity of antiapoptotic factors allows malignant cells to keep growing. As summarized in Supplementary Table IV to be found online at http://informahealthcare.com/doi/abs/10.3109/07853890.2014.892296 and Figure 4, the genes encoding pro- and anti-apoptotic proteins exhibit circadian rhythmicity; thus, the circadian system might help maintain the right balance between pro and antiapoptotic signals (124). In addition, some proteins of the cell cycle and apoptotic pathways have a dual role; following stimulation they induce the cell cycle, cell proliferation, or cell death depending on the cellular context.

Apoptosis can be initiated by two different pathways, the extrinsic (death receptor) and intrinsic pathways (124). The extrinsic, mitochondria-independent pathway requires the interaction of extracellular ligands with cell death receptors from the TNF-receptor family; this results in the clustering of the receptors and the cytoplasmic adaptor molecules, finally leading to the activation of pro-caspase 8 to caspase 8. The intrinsic pathway can be triggered by a wide group of nonreceptormediated stimuli that induce cellular damage, such a radiation, toxins, hypoxia, and free radicals, among others. These stimuli cause changes that result in the formation and opening of the mitochondrial permeability transition pore, the loss of mitochondrial transmembrane potential, and the release of proapoptotic proteins from the intermembrane space of the mitochondria into the cytosol. The proteins activate pro-caspase 9, resulting in an active enzyme. These processes are regulated by a wide range of pro- and anti-apoptotic regulatory proteins from the Bcl-2 family. Initiator caspases (caspase-2, 8, 9, or 10) activate the execution caspases, in particular the caspase-3. This caspase and other execution caspases function as the effectors of apoptosis by cleaving various substrates.

The data summarized in Figure 4 demonstrate that both the extrinsic and intrinsic pathways contain many proteins whose genes exhibit circadian rhythmicity. One of the highest incidences of circadian rhythmicity in the studied tissues belongs to the gene Casp3, which encodes the executioner caspase-3, even if many other genes encoding the proteins of the extrinsic (e.g. Tnfsf12, Tnfsf10, Casp8) and intrinsic pathways (genes encoding the proteins of Bcl-2 family) also exhibit circadian rhythmicity in many tissues. The meta-analysis showed circadian rhythmicity of many genes encoding proteins of Bcl-2 family: the pro-apoptotic genes encoding proteins essential for the initiation of apoptosis (e.g. Bid, Bcl2l11(Bim), Bik) and for opening of the mitochondrial pores (e.g. Bak1, Bax) and the anti-apoptotic genes (e.g. Bcl2, Bcl2l1(Bclx)). Similarly, the expression of the BCL2 and BAX proteins significantly varies during a 24-hour period in bone marrow (126).

Direct connection between the core clock genes and the apoptotic genes of the extrinsic and intrinsic pathways has been shown in some recent studies. The clock protein CRY participated in the extrinsic TNFα-dependent pathway (127) and PER1 in the intrinsic pathway (81,128,129). Per1 knockdown was associated with the upregulation of BAX and the downregulation of BCL-2 in hepatocellular carcinoma cells and the overexpression of PER2 in lung, mammary, and pancreatic carcinoma cell lines downregulated the expression of genes Bcl-X and Bcl2 and upregulated the expression of Bax gene; similar changes were observed at the levels of corresponding proteins.

Another avenue of cross-talk between the circadian clock and apoptotic pathways represent the transcription factors DEC1 and DEC2, which are involved in the core circadian clock; they repress CLOCK:BMAL1-induced transactivation of the Per1 promoter. Using human breast cancer cells, it has been shown that DEC1 acts in a pro-apoptotic and DEC2 in an anti-apoptotic manner (130,131). Knockdown of DEC2 stimulated the transcript and protein levels of both the extrinsic (Fas, Casp8) and the intrinsic pathways (Bax), whereas overexpression of DEC2 inhibited pro-apoptotic factor BIM and reduced the amount of caspase-8. Other links that might interconnect the circadian clock with the cell cycle and apoptosis are the proteins p53 and GADD45, which are expressed rhythmically in several tissues (Figure 1). Cellular stress, such as DNA damage or oncogene expression, leads to stabilization and activation of these tumor suppressors. Depending on the cellular context, the activation results in one of two different outcomes: the cell cycle arrest (see above) or apoptotic cell death (79). In contrast to hepatocytes of the wild-type mice, the hepatocytes of Clock mutant mice have decreased expression of apoptosis-inducing factors, and the response to cytotoxic stress is not associated with accumulation of p53 and caspase-3/7 (132). Additionally, GADD45 proteins are able to activate apoptosis via the JNK and/or p38 signaling pathways without induction of p53 (133).

Conclusions

A number of studies have indicated that an unhealthy lifestyle leading to the disruption of circadian homeostasis is a risk factor for tumorigenesis. Cell cycle, proliferation, and apoptosis are closely intertwined with the circadian clock; thus, the disruption of circadian rhythms appears to be linked to tumorigenesis and carcinogenesis. Because tumors are derived from numerous tissues with various etiologies and tumor progression is associated with an endless combination of genetic and epigenetic alterations, it is evident that the molecular clock may play a role in the fine-tuning processes associated with tumor growth rather than act as a determinant of cell growth and differentiation. Several lines of evidence suggest that the intrinsic oscillatory mechanism can influence the regulation of the timing and efficiency of cell cycle events in proliferating tissues, and the deregulation of this process has been observed in tumors. Our meta-analysis shows that the possible relationship between the circadian core clock and tumorigenesis cannot be limited to only regulation by WEE1, as overall circadian variations were found in the expression of many other genes associated with cell growth and tumor promotion. It would be very interesting to unravel whether the circadian expression of these genes is really affected in specific tumors and whether these changes play causal roles in tumorigenesis. These findings will increase our knowledge of neoplastic transformation and tumor growth, as well as impact new strategies for cancer prevention and therapy.

Declaration of interest: The work in the authors’ laboratory has been supported by the Czech Science Foundation (grant 13-08304S). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.