Circadian clocks in health and disease: Dissecting the roles of the biological pacemaker in cancer

Cells regulate their growth and division using cell cycle checkpoints, which ensure timely progression of the cell cycle under normal physiological conditions, and also halt cell cycle progression in instances of DNA damage, erroneous mitosis, or environmental stressors ( Collins et al., 1997; Kastan & Bartek, 2004). The duration and transition of each cell cycle phase is orchestrated by the activation of specific cyclin-dependent kinases (CDKs) by their respective cyclins ( Graña & Reddy, 1995; Vermeulen et al., 2003). Whereas expression of CDKs remains relatively constant throughout the cell cycle, each cyclin peaks in expression in a staggered and coordinated manner to drive the cell cycle with appropriate timing ( Graña & Reddy, 1995; Vermeulen et al., 2003). Given the rhythmic nature of both the cell cycle and the circadian clock, several studies have worked to investigate and define the connections between these two systems. It has been suggested that circadian rhythms and the cell cycle are tightly phase-coupled and oscillate with a 1:1 frequency in mouse fibroblasts ( Feillet et al., 2014), and this synchronization has even been shown at the single cell level in mammalian NIH3T3 fibroblasts ( Bieler et al., 2014). More recently, data suggests that the cell cycle and the circadian clock can synchronize each other bidirectionally in mammalian systems ( Yan & Goldbeter, 2019). However, the extent of this coupling is still not well understood, as some studies report additional findings that the cell cycle and circadian clock can in fact operate independently, as demonstrated in Rat-1 fibroblasts as well as Lewis lung carcinoma cell lines ( Yeom et al., 2010; Pendergast et al., 2010).

Given the intimate links between the circadian clock and the cell cycle, what are the molecular connections driving this interplay? Importantly, several studies have investigated connections between the circadian clock and the proto-oncogene MYC, a master regulator of cell cycle control that promotes cellular growth by driving cyclin expression and repressing CDK inhibitor activity ( Burchett et al., 2021). In regard to transcriptional regulation of c-Myc , it has been reported that Bmal1 ^-/- mice exhibited increased expression of c-Myc , whereas c-Myc expression was decreased in Cry1 ^-/-;Cry2 ^-/- mice ( Liu et al., 2020), demonstrating a strong correlation between these networks. Additionally, MYC and NMYC can impinge on circadian rhythms in U2OS and SHEP cells, respectively, further highlighting the molecular crosstalk between the clock and the cell cycle that also integrates with cellular metabolic state ( Altman et al., 2015; Shostak et al., 2016; Moreno-Smith et al., 2021). In regard to regulation of MYC protein levels, it was demonstrated that CRY2 mediates MYC degradation and that Cry2 ^-/- knockout induced increased proliferation and transformation rates ( Huber et al., 2016).

In addition to circadian links to MYC, clock-dependent transcription has been observed for many other cell cycle modulators, including cyclin D, cyclin E, cyclin A, and cyclin B in human epithelium ( Bjarnason et al., 1999; Fu et al., 2002). Likewise, G2/M regulator WEE1, which inhibits Cyclin B1-CDK1 activity, has oscillating protein expression and kinase activity and this oscillation was dampened in Cry1 ^-/-;Cry2 ^-/- mice ( Gréchez-Cassiau et al., 2008; Matsuo et al., 2003). Another study found that CLOCK and BMAL1 knockdown leads to the suppression of WEE1 and thus an increased activation of apoptosis in human hepatocellular carcinoma cell lines, again confirming circadian influence on WEE1 regulation ( Qu et al., 2023). Additional CDK inhibitors such as p21 ^cip1/waf1 and p16-Ink4A also oscillate under circadian control and this rhythmicity was lost in Bmal1 ^-/- mice or Per1 ^Brdm1/Brdm1;Per2 ^Brdm1/Brdm1 mutant mice, respectively ( Gréchez-Cassiau et al., 2008; Kowalska et al., 2013). Taken together, these data highlight the important molecular links between the circadian clock and cell cycle control mechanisms.

Due to a multitude of interactions between circadian proteins and cell cycle checkpoints and drivers, it is not surprising that circadian rhythm disruption can modify rates of cellular proliferation. For example, genetic clock disruption in mouse osteoblasts via Per1 ^-/-;Per2 ^m/m knockout or Cry1 ^-/-;Cry2 ^-/- knockout resulted in increased proliferation ( Fu et al., 2005). Circadian clock disruption can potentially interfere with normal rates of cellular growth, introducing susceptibility to disease and cancer. Altogether, this data suggests that oscillations of clock proteins contribute to the proper expression of important cell cycle regulators that impact cellular proliferation.

Another important aspect of cell cycle regulation features the DNA damage response (DDR). Individual cells can receive tens of thousands of DNA lesions per day, which leads to replication errors, transcription blockage, and even permanent mutations if left unrepaired ( Jackson & Bartek, 2009). Thus, the DDR has evolved to preserve genome integrity by recognizing various forms of DNA damage, stimulating DNA repair, inhibiting cell cycle progression until the repair is complete, and inducing apoptosis if the damage is irreparable ( Giglia-Mari et al., 2011; Jackson & Bartek, 2009; Roos & Kaina, 2013).

In mammals, central DDR proteins ATR and ATM instigate the DDR by phosphorylating CHK1 and CHK2, respectively ( Jackson & Bartek, 2009). Consequently, phosphorylated CHK1 and CHK2 activate transcription factor p53 to halt cell cycle progression ( Ronco et al., 2017). It was reported that circadian proteins CRY1 and TIM modulate ATR and CHK1 activity in a time-of day dependent fashion, although it was found that Cry1 ^-/-;Cry2 ^-/- mouse embryonic fibroblasts (MEFs) still retained appropriate levels of checkpoint activity ( Kang & Leem, 2014). Also, ATM and CHK2 form a complex with circadian protein PER1, and it was further demonstrated that PER1 knockdown reduced ATM-mediated CHK2 phosphorylation and dampened apoptotic response to DNA damage in human colon cancer cell line HCT116 ( Gery et al., 2006).

Furthermore, a bi-directional crosstalk between the circadian clock and p53 has been reported. The p53 tumor suppressor plays a key role in stimulating DNA damage checkpoints and a circadian oscillation of p53 transcription has been reported in human oral epithelium ( Bjarnason et al., 1999). Concurrently, it has been demonstrated that p53 modulates circadian activity in mice by directly binding to the promoter of Per2 and repressing Per2 expression ( Miki et al., 2013). Moreover, PER2 directly binds to the C-terminal end of human p53 and slows MDM2-mediated degradation of p53 ( Gotoh et al., 2014), indicating multiple points of interplay between these two mechanisms. The consequences of clock regulation on p53 were evident in Per2 ^m/m mutant mice that exhibited lower levels of p53, increased resistance to p53-mediated apoptosis, and higher sensitivity to γ radiation ( Fu et al., 2002). p53 activates the CDK inhibitor p21 ^cip1/waf1 to induce cell cycle arrest ( Al Bitar & Gali-Muhtasib, 2019). As stated in the previous section, p21 ^cip1/waf1 expression oscillates under circadian control, further demonstrating clock regulation of the DDR. Interestingly, it was reported that either Cry1 ^-/- or Cry2 ^-/- MEFs exhibit altered expression patterns of the p21 ^cip1/waf1 transcript, Cdkn1a, in response to genotoxic stress via doxorubicin ( Papp et al., 2015). This study further demonstrated that genotoxic stress can shift the CRY1/CRY2 ratio and consequently change circadian period length ( Papp et al., 2015), again demonstrating the crosstalk between the DDR and the molecular machinery of the circadian clock.

In summary, these studies suggest that circadian proteins exert a wide influence on proper cellular response to the daily insults of DNA damage. Impaired DDR via clock disruption increases the likelihood of cell proliferation despite unresolved mutations, which is a major contributor to cancer progression. Furthermore, since DDR proteins are commonly targeted during chemotherapy to inhibit rapidly dividing cells, understanding the effects of oscillating circadian proteins and their impact on the DDR may result in enhanced efficacy of cancer therapeutics.

The circadian clock not only plays a role in regulating DNA damage checkpoints, but also affects the ability of cells to perform DNA repair. Cells are equipped with multiple repair pathways that act to maintain DNA sequence fidelity following damage from endogenous and exogenous sources. Interestingly, while each DNA repair pathway has several components, the activities of certain key components exhibit striking transcriptional regulation through the circadian clock ( Sancar et al., 2010).

Nucleotide excision repair (NER) removes bulky chemical adducts that distort the DNA helix, most importantly UV-induced intrastrand crosslinks ( Marteijn et al., 2014). NER relies on the xeroderma pigmentosum group A (XPA) protein to recognize the lesion and coordinate incision and removal by the XPF-ERCC1 endonuclease complex ( Marteijn et al., 2014). It was recently described that XPA oscillates in a circadian fashion, thus repair activity after UV irradiation also follows circadian rhythmicity. However, this rhythmicity was lost in Cry1 ^-/-;Cry2 ^-/- mice, clearly demonstrating that NER activity is regulated by the clock ( Gaddameedhi et al., 2011). As sunlight is the major source of UV radiation, this connection between DNA repair of UV-induced damage and the circadian clock is intriguing.

Base excision repair (BER) uses a variety of different DNA glycosylases to remove damaged bases, leaving an abasic site that is subsequently processed by enzymes that carry out cleavage, gap-filling and ligation to restore DNA integrity ( Krokan & Bjørås, 2013). The expression and activity of one such glycosylase, 8-Oxoguanine DNA glycosylase (OGG1), oscillates under clock control and OGG1 levels were disrupted in a human cohort performing shift work ( Manzella et al., 2015). Importantly, this study was performed using lymphocytes collected from human blood whereas another study found that OGG1 does not oscillate in human keratinocytes ( Hettwer et al., 2020). This suggests a potential cell-type specific role of OGG1 circadian regulation. As OGG1 recognizes a specific type of oxidative damage (i.e., 7,8-dihydro-8-oxoguanine opposite cytosine or thymine), it remains to be seen why this enzyme has evolved to be clock-controlled. One interesting link is that OGG1 has been shown to be required to prevent mutations induced by UVA ( Kozmin et al., 2005), suggesting an additional role of the clock in repairing DNA damage following sunlight exposure.

DNA double strand breaks (DSBs) represent one of the most serious threats to genome integrity and multiple repair pathways have evolved for their repair, including homologous recombination (HR) and non-homologous end joining (NHEJ) ( Chang et al., 2017; Sterrenberg et al., 2022). Due to the number of environmental and cellular sources of DSBs, understanding the role of the circadian clock in their repair is critical. Using HEK293T cells, CLOCK binding was found at several enhancer or transcriptional regulatory sites controlling DNA damage related genes including CDKN1A encoding p21, which mediates cell cycle arrest, as well as BRCA1 and RAD50, which play important roles in DSB repair ( Alhopuro et al., 2010). Furthermore, CLOCK knockdown in human U2OS osteosarcoma cells resulted in abnormal cell cycle checkpoint response following irradiation and increased sensitivity to mitomycin C, indicative of a CLOCK-dependent response to repair DSBs ( Cotta-Ramusino et al., 2011). CLOCK was also found to localize to laser-induced DSBs in U2OS cells, suggesting a potential direct role in the cellular signaling machinery required for DSB repair ( Cotta-Ramusino et al., 2011). In addition to CLOCK, CRY1 is another circadian protein linked to DSB repair efficiency. CRY1 knockdown showed delayed DSB resolution in C4-2 and 22Rv1 cell cultures, and conversely, DSB resolution is enhanced upon treatment with the CRY1 stabilizer KL001 ( Shafi et al., 2021). Transcriptomic analysis suggested that CRY1 regulated the expression of several major HR genes (including RAD51, BRCA1, and BRCA2) and other genes involved in NER, BER, mismatch repair (MMR), and NHEJ ( Shafi et al., 2021). This study was specifically carried out in human prostate cancer cell lines as well as tissues from prostate cancer patients, and further studies are needed to define the role of CRY1 in regulation of DNA repair genes in other tissues, particularly those that are not hormone responsive.

Overall, these studies show that the effect of the circadian clock on DNA repair is widespread across multiple repair pathways. Although the mechanistic links continue to be investigated, current data suggests that robust circadian rhythms contribute to optimal genome integrity. Further understanding of how the circadian clock potentiates faithful DNA repair through multiple pathways is paramount for developing strategies to both prevent cancer, and to establish better and less toxic treatments for patients undergoing chemo- and radiation- therapy that acts through damaging DNA.

The goal of the immune system is to be primed to respond to insult through a complex network of different organs, proteins and pathways. It may be advantageous for immune parameters to cycle with activity of an organism, potentially allowing for the host to respond more efficiently to infection. In support of this, the circadian clock has been shown to regulate key parameters of immunity including cytokine release, immune cell number and trafficking, as well as the inflammatory response.

Immune cells, including splenic macrophages, dendritic cells (DCs), and B cells have been found to have cell-autonomous circadian clocks which directly control cellular immune function and timing ( Keller et al., 2009; Silver et al., 2012). Cytokines and chemokines are small proteins that regulate the growth, activity and trafficking of immune cells and proper regulation of these proteins is essential for host immune defense. Importantly, the circadian clock has been linked to the production and release of cytokines and chemokines. Upon bacterial endotoxin stimulation, the secretion of TNFα and IL-6 by isolated ex vivo spleen derived macrophages was found to oscillate in a time-of-day dependent manner, including 8% of the macrophage transcriptome ( Keller et al., 2009). Additional studies demonstrated an important role for Bmal1 in regulating cytokine response. Temporal gating of endotoxin-induced cytokine response in mice, a crucial feature of innate immunity, is dependent on the circadian clock as rhythmic gating of endotoxin response is lost in Bmal1^-/- macrophages ( Gibbs et al., 2012). This was found to be due to the suppression of Nr1d1, hereafter referred to as Rev-Erbα ( Gibbs et al., 2012). An additional role of Bmal1 in regulating the immune response was identified with the genetic ablation of Bmal1 in bronchiolar cells that disrupted the rhythmic expression of the CXCL5 chemokine ( Gibbs et al., 2014). These data suggest that the rhythmic release of cytokines is directly regulated by the circadian clock.

In addition to the rhythmic secretion of cytokines by immune cells, the circadian clock controls immune cell number and infiltration. For example, the number of hematopoietic stem cells (HSCs) and mature immune cells released from the bone marrow into the blood peaks at the beginning of the rest phase in mice ( Méndez-Ferrer et al., 2008). In addition to the release of immune cells into the bloodstream, the circadian clock also modulates immune cell trafficking into tissues as evidenced by the rhythmic expression of CXCL5 and CXCL12 that regulate the trafficking and infiltration of neutrophils and HSCs, respectively ( Gibbs et al., 2014; Méndez-Ferrer et al., 2008). Human studies provide additional evidence of circadian regulation of immune cell trafficking. Immune cells present in the blood of individuals on a normal sleep-wake cycle were compared to those on 24 hours of wakefulness ( Dimitrov et al., 2007). It was found that the number of DCs and T cells in the blood is highly rhythmic and that sleep induced the expression of IL-12 which increased the number of monocytic DCs in the blood ( Dimitrov et al., 2007). A more recent study found that individuals with blunted rest-activity rhythms exhibited increased inflammatory markers and elevated circulating white blood cells and neutrophils ( Xu et al., 2022). These studies demonstrate a clock-controlled immune response through regulation of immune cell release into the bloodstream and trafficking into tissues.

Clock control of the immune system is critical for proper response to infection ( Kiessling et al., 2017) and disease ( Gibbs et al., 2014; Kitchen et al., 2020), and even vaccination ( Cervantes-Silva et al., 2022). In support of this, mice infected with Salmonella enterica in the early rest period exhibited a high pathogen load and a stronger proinflammatory response ( Bellet et al., 2013) and the magnitude of Leishmania parasitic infection in mice varied over 24 hours ( Kiessling et al., 2017). These differences in infection and inflammation may be due to the time-dependent release of cytokines and immune cells. Indeed, the circadian expression of chemo attractants and the rhythmic infiltration of neutrophils and macrophages was lost in clock deficient macrophages ( Kiessling et al., 2017; Sato et al., 2014). Additionally, pulmonary inflammation was found to be regulated by the rhythmic expression of the chemokine CXCL5 leading to time-of-day dependent neutrophil recruitment to the lung ( Gibbs et al., 2014). Bmal1^-/- bronchiolar cells lack this rhythmic CXCL5 expression leading to exaggerated inflammatory response and an impaired host response to Streptococcus pneumoniae infection ( Gibbs et al., 2014). Bmal1 deletion suppresses Rev-erbα expression, and it was found that Rev-erbα ^-/- mice exhibit an exaggerated neutrophilic inflammatory response ( Pariollaud et al., 2018). Furthermore, myeloid specific deletion of Bmal1 disrupts the diurnal trafficking of Ly6Hi inflammatory monocytes and promotes inflammation by inducing expression of monocyte attracting chemokines ( Nguyen et al., 2013).

Overall, these studies establish the circadian clock as a critical regulator of the immune response through the release of cytokines and the trafficking of immune cells. This leads to a time-of-day dependent proinflammatory response to challenge such as bacterial or pathogenic infection. Moreover, disruption of the circadian clock has the potential to alter the daily rhythm of the immune system and lead to various types of diseases, including cancer.

About one quarter of the US population participates in night shift work ( Alterman et al., 2013; Drake & Wright, 2017), which causes significant misalignment between the endogenous circadian clock and the sleep-wake cycle ( James et al., 2017). Night shift work has been implicated as a risk factor for cancer and a systematic review of night shift work and cancer was recently reported ( IARC, 2020). Several studies were identified that aimed to assess a correlation between night shift work and cancer risk, and these reports are summarized in Table 1. The most extensively studied association was between night shift work and breast cancer, and the majority of these reports found that night shift work increased the risk of developing breast cancer ( Cordina-Duverger et al., 2018; Hansen & Lassen, 2012; Jones et al., 2019; Schernhammer et al., 2006). Several studies also noted an increased risk with duration of exposure and cumulative exposure to night shift work ( Cordina-Duverger et al., 2018; Davis et al., 2001; Hansen & Lassen, 2012; Hansen & Stevens, 2012; Lie et al., 2011). In addition to breast cancer, shift work was also found to increase the risk of developing prostate, colon and rectum, lung, stomach, ovarian and pancreatic cancer. Although numerous studies cite a positive correlation between night shift work and cancer incidence, other studies report no effect ( Jones et al., 2019; Koppes et al., 2014; Li et al., 2015; O’Leary et al., 2006; Pronk et al., 2010; Travis et al., 2016; Vistisen et al., 2017). There are multiple explanations for the contradictory data, including the lack of a standardized definition for night shift work, self-reporting collection process, and adjustment for confounding factors such as socioeconomic status and lifestyle. These limitations should be addressed and larger, more comprehensive studies are needed with multiple cancer types to define the epidemiological link between the circadian clock and cancer risk.

The previous section highlighted the potential increase in cancer incidence in populations that participate in night shift work, which is known to disrupt circadian rhythms. However, there is mounting concern for circadian disruption in the general population as the access to technological devices continues to increase. Gradisar et al. demonstrated that nine out of 10 individuals surveyed use a technological device in the hour before bed, with the use increasing in individuals under 30 years of age ( Gradisar et al., 2013). Among the Japanese population, young adults between the ages of 15 to 20, were exposed to the highest intensity of artificial light-at-night ( Chen et al., 2022). The exposure to dim light at night through the use of devices has been shown to disrupt circadian rhythmicity by suppressing melatonin and impairing sleep quality ( Lee & Kim, 2019). This suggests that younger individuals may be exposed to more environmental factors that disrupt the circadian clock than older populations. Strikingly, the average annual increase in the incidence of all cancers in young adults aged 15 to 39 years old has continued to increase since 1975 ( Miller et al., 2020). A review of 98 articles published between 1995–2020 found that the incidence of colorectal, breast, kidney, pancreas, and uterine cancer is increasing in younger age groups ( di Martino et al., 2022). In addition to the increasing incidence, studies have also suggested that the underlying biology of cancer in young adults differs from the same cancer in children or older individuals ( Tricoli et al., 2016, 2018). Altogether, this introduces the idea that environmental circadian clock disruption in younger populations may contribute to the increasing incidence of early-onset cancers, though further studies are needed to confirm this experimentally.

It is worth noting that the increasing trends of early-onset cancers are strongest for colorectal cancer (CRC). Between 1975 and 2010, there has been a steady decline in CRC incidence rates in adults over the age of 50. However, in patients aged 20 to 34, the incidence of CRC has continued to rise ( Bailey et al., 2015). If this trend continues, it is expected that by 2030, the incidence of colon and rectal cancer in individuals aged 20 to 34 will increase by 90% and 124.2%, respectively ( Bailey et al., 2015). It was also identified that the increasing incidence of CRC in younger populations was greatest among Hispanics and African Americans, suggesting an alarming cancer health disparity ( Augustus & Ellis, 2018; Muller et al., 2021; Singh et al., 2014).

Importantly, the intestine may be particularly sensitive to circadian disruption due to several reasons. The intestine is a highly regenerative organ, with complete cell renewal occurring every few days ( Van Der Flier & Clevers, 2009). This constant renewal process is tightly regulated by the circadian clock, and disruption of circadian rhythms can affect the timing and coordination of this turnover process ( Codoñer-Franch & Gombert, 2018; Stokes et al., 2017; Yoshida et al., 2015). The gut microbiota, which play a crucial role in digestion and immune function, also exhibit circadian rhythmicity and can be negatively impacted by circadian disruption ( Heddes et al., 2022; Leone et al., 2015; Liang et al., 2015; Thaiss et al., 2016). Moreover, food intake is a powerful environmental cue synchronizing the circadian clock in peripheral tissues ( Zarrinpar et al., 2014). Feeding mice only during the light phase, when mice are inactive, causes a phase shift in peripheral clocks of the liver, kidney, heart and pancreas ( Damiola et al., 2000; Stokkan et al., 2001), demonstrating that the timing of food intake can disrupt the circadian clock. Nutritional challenge has also been shown to dynamically impact the circadian clock, including high fat diet and time-restricted eating ( Acosta-Rodríguez et al., 2022; Chaix et al., 2019; Eckel-Mahan et al., 2013; Hatori et al., 2012). These studies establish the importance of coordinated cell renewal, gut microbiota, diet and timing of food intake in maintaining robust circadian rhythms in the intestine. It will be important to study the impact of alterations in these environmental and behavioral factors on the alarming increase in CRC rates in younger populations, as well as other cancer types.

Although there is mounting evidence suggesting that the circadian clock is implicated in various types of cancer, the mechanism underlying the role of the clock in cancer is still being uncovered. Table 2 outlines significant findings that provide clues for how the circadian clock functions in various cancer types and model systems. Based on these studies, the circadian clock has been implicated in both the initiation and the progression of cancer through the regulation of oncogenic pathways, cell cycle control, DNA damage repair, stemness, immunity and metastasis ( Figure 2). However, the effect of clock disruption on tumorigenesis may be tissue and model-specific. For example, knock out of the core clock gene BMAL1 in mouse models of solid tumors promotes tumor progression in CRC ( Chun, Fortin, Fellows et al., 2022; Stokes et al., 2021), lung ( Papagiannakopoulos et al., 2016), and other cancer types ( Lee et al., 2010) but reduces the development of cutaneous squamous tumors ( Janich et al., 2011). Furthermore, downregulation of BMAL1 in human glioblastoma stem cells halts their growth ( Dong et al., 2019; Puram et al., 2016). As more studies are done in multiple cancer types and model systems, we may begin to better delineate the tissue-specific effects of clock disruption on cancer.

Wnt signaling is an important pathway for many processes including development, proliferation, and apoptosis ( Nusse & Clevers, 2017). This is especially relevant for the intestine, a highly regenerative organ where differentiated cells are replaced every four to five days by stem cells located in the crypt base ( Van Der Flier & Clevers, 2009). The Wnt pathway is upregulated when Wnt ligands bind the frizzled receptor and trigger inactivation of the destruction complex, composed of APC, GSK3-β and AXIN ( Neufeld et al., 2000; Orford et al., 1997; Rubinfeld et al., 1993; Su et al., 1993). As a result, β-catenin evades proteosome-dependent degradation, and shuttles into the nucleus to co-activate TCF-LEF mediated transcription of Wnt-dependent genes ( Hoverter et al., 2014; Molenaar et al., 1996).

The intestine is a highly rhythmic organ and rhythmicity is important in functions such as peristalsis, permeability and secretion of digestive enzymes ( Codoñer-Franch & Gombert, 2018). The molecular clock is involved in regulating intestinal circadian rhythms as core clock genes are expressed in many intestinal cell types including stem, progenitor, tuft, enteroendocrine and enterocytes ( Habowski et al., 2020). Circadian rhythms of intestinal stem cells (ISCs) are thought to be driven, at least in part, by Wnt signaling secreted from differentiated cells. ISC clocks were responsive to both Wnt and Hippo signaling in the stem cell niche ( Parasram et al., 2018). Furthermore, Paneth cells were found to rhythmically secrete Wnt and many Wnt pathway components have rhythmic expression ( Matsu-Ura et al., 2016; Soták et al., 2013). This is likely to be essential for proper ISC function as Wnt signaling was found to couple the clock and the cell cycle in the intestine ( Matsu-Ura et al., 2016) and the coupling of clock and cell cycle is conserved across multiple species ( Hong et al., 2014a; Yang et al., 2010). In the intestine, the molecular clock was found to gate cell cycle progression and was important for ISC regeneration after DSS induced damage ( Karpowicz et al., 2013; Matsu-Ura et al., 2016).

The Wnt signaling pathway is highly mutated in human CRC with nearly all tumors containing inactivating mutations in APC or GSK3-β, or stabilizing mutations in CTNNB1 (β-catenin) ( Kwong & Dove, 2009). Around 80% of sporadic human CRC contain a mutation in APC ( Fearnhead et al., 2001). In many cases this is followed by mutation of the wild type allele, also known as loss of heterozygosity (LOH) ( Fearnhead et al., 2001; Kwong & Dove, 2009). Due to its important role in the destruction complex, APC mutation results in aberrant activation of the Wnt signaling pathway ( Korinek, 1997; Morin, 1997; Moser et al., 1990). APC mutations are sufficient to drive CRC initiation of adenoma growth ( Cheung et al., 2010; Lamlum et al., 2000; Rowan et al., 2000; Zauber et al., 2016; Zhang & Shay, 2017). However, secondary driver mutations in key genes such as KRAS, TP53, and SMAD4 are required for progression to adenocarcinoma ( Drost et al., 2015; Fearon & Vogelstein, 1990; Vogelstein et al., 1988). The circadian clock has recently been identified as a secondary driver of CRC. Bmal1 loss was found to promote Apc LOH by increasing genome instability and resulting in Wnt signaling hyperactivation in mice ( Chun, Fortin, Fellows et al., 2022). Additionally, downregulation of PER2 in human colon cancer cells HCT116 and HT29 increased β-catenin levels and cell proliferation ( Wood et al., 2008). Increased β-catenin in CRC cell lines also enhanced PER2 degradation by upregulating β-TrCP, an E3 ubiquitin ligase component ( Yang et al., 2009). As APC is involved in regulation of cell-cell adhesion, microtubule stability, cell cycle and apoptosis ( Fearnhead et al., 2001), clock disruption mediated APC LOH could perturb multiple pathways important in CRC progression.

Aside from Wnt signaling, other pathways have also been implicated in clock mediated acceleration of CRC. The Hippo pathway regulates multiple key processes including proliferation, differentiation, tissue growth, and regeneration. A cascade of serine/threonine kinases act to sequester Yes associated protein (YAP1) and transcriptional activator with PDZ binding motif (TAZ) in the cytoplasm and prevent them from activating the pro-survival TEA DNA binding (TEAD) family of transcription factors. Dysregulation results in an increase in YAP/TAZ which is associated with many human cancers, mediating increased proliferation and metastasis ( Calses et al., 2019). In an Apc^Min/+ mutant mouse model, YAP1 was found to be required for the progression of early initiating cells by suppressing differentiation and promoting regeneration ( Gregorieff et al., 2015). The Hippo pathway may be involved in clock mediated acceleration of CRC. Yap and Tead4 increased in Apc^Min/+; Bmal1^-/- mice and were associated with increased self-renewal ( Stokes et al., 2021). Furthermore, Apc^ex1-15/+; Bmal1^-/- organoids had increased expression of YAP/TAZ pathway components compared to Apc^ex1-15/+ organoids ( Chun, Fortin, Fellows et al., 2022). In summary, clock disruption accelerates the pathogenesis of CRC, and based on data from pre-clinical studies, the circadian clock likely impinges on several important signaling pathways that regulate intestinal biology.

Misregulation of molecular clock components have frequently been identified in human CRC. Multiple studies have reported decreased BMAL1, CRY1-2 and PER1-3, and increased CLOCK, CSNK1E and TIM in tumor tissue relative to matched healthy mucosa ( Hong et al., 2014b; Krugluger et al., 2007; Mazzoccoli et al., 2011, 2016; Oshima et al., 2011; Zeng et al., 2014). This was also linked to disease progression as reduced BMAL1, PER1 or PER3 expression was associated with poor overall survival ( Mazzoccoli et al., 2011; Zeng et al., 2014). Additionally, clock genes were found to be mutated in cancer and therefore might be involved in pathogenesis. A large fraction of CRC patients with microsatellite instability (MSI) had a mutation in CLOCK which could decrease CLOCK expression in MSI CRC cell lines ( Alhopuro et al., 2010; Mazzoccoli et al., 2011).

Pre-clinical genetic mouse models have also demonstrated that clock disruption accelerates CRC pathogenesis. In the azoxymethane and dextran sodium sulfate (AOM/DSS) model of colitis associated CRC, the circadian rhythmicity of Per1, Per2, Reverb, Dbp and Bmal1 was reduced in tumors compared to healthy colon ( Soták et al., 2013). In an Apc ^Min/+ model of CRC, tumors exhibited reduced overall expression of key clock components Rev-Erbα, Bmal1 and Per2, with complete loss of Per2 rhythm ( Stokes et al., 2021; Yang et al., 2009). Furthermore, clock disruption was found to increase tumor burden when Per2 or Bmal1 were deleted in Apc ^Min/+ mice ( Stokes et al., 2021; Wood et al., 2008). In a novel mouse model, where exons 1 to 15 in Apc were deleted in one allele ( Apc ^ex1-15/+), Bmal1 knockout increased tumor incidence, enlarged polyps and decreased survival ( Chun, Fortin, Fellows et al., 2022). Additionally, environmental circadian disruption through use of a light shift paradigm or constant light increased both polyp formation and size of tumors in Apc ^ex1-15/+ and Apc ^min/+ mice ( Chun, Fortin, Fellows et al., 2022; Stokes et al., 2021). Together these results suggest that circadian disruption can play a key role in driving pathogenesis of CRC.

Promoting robust circadian rhythms through consistent sleep and feeding behavior is an important regulator of physiological health. However, night shift workers are faced with irregular activity-rest and feeding-fasting rhythms as well as artificial light at night exposure, all of which are known to disrupt the circadian clock. Key literature was reviewed above that aimed to define the correlation between night shift work and cancer prevalence. Although this body of literature requires more comprehensive studies to draw definitive conclusions, the importance of proper alignment of circadian rhythms has emerged as a key theme. Numerous studies cite a significant increase in cancer risk after long-term night shift work, typically 15–20 years ( Åkerstedt et al., 2015; Hansen & Lassen, 2012; Wegrzyn et al., 2017). Risk was also seen to increase with duration of exposure ( Davis et al., 2001; Hansen & Lassen, 2012; Hansen & Stevens, 2012; Lie et al., 2011). For example, in nurses who worked night shift for over five years, the risk of developing breast cancer increased from an odds ratio of 1.4 to 1.8 with increasing consecutive night shifts ( Lie et al., 2011). In order to reduce the risk of cancer in night shift workers, the consecutive duration of night shifts may need to be limited as well as the cumulative exposure to night shift work.

Circadian misalignment, defined as a disruption or misalignment between an individual’s internal circadian clock and the external cues such as light-dark and feeding-fasting cycles, is an unavoidable consequence of night shift work. However, recent research has recommended lifestyle interventions as a means of combating these effects. Night shift workers often disrupt their feeding-fasting patterns which has been shown to disrupt glucose metabolism ( Spiegel et al., 1999). Night shift work has also been significantly associated with metabolic syndrome ( Wang et al., 2014), which increases the risk for developing various types of cancer ( Esposito et al., 2012). Therefore, increasing metabolic health through lifestyle intervention may combat the increased risk of cancer. One such intervention is time-restricted eating (TRE), which involves limiting the eating window to 6–12 hours per day ( Manoogian et al., 2022). In a study with prediabetic men, limiting the feeding window to six hours for five weeks improved insulin sensitivity, blood pressure, oxidative stress, and appetite ( Sutton et al., 2018). A recent study found that a 10-hour feeding window in 24-hour shift workers is a feasible intervention to reduce weight, improve cardiometabolic health, sleep quality, and mood ( Manoogian et al., 2022).

It has been well established that night shift is associated with circadian misalignment, however, the general population is increasingly at risk of circadian misalignment through inconsistent eating patterns and artificial light-at-night exposure. Therefore, it may be beneficial to update cancer screening measures ( Patel et al., 2022; Wolf et al., 2018). As the percentage of early-onset cancer increases, the screening age should decrease accordingly for early detection and prevention measures. Promoting robust circadian rhythms through consistent sleep, feeding, regulation of night shift work, and lifestyle interventions such as TRE may help improve parameters that impinge on human health and could offset the impacts of circadian misalignment on specific cancer types.

Cancer chronotherapy refers to the timing of an anticancer drug to increase efficacy and decrease toxicity. This approach is based on the rationale that the drug will be better tolerated at certain times of day based on the mechanism of action of the drug. A recent comprehensive review of chronomodulated chemotherapy has recently been reported ( Printezi et al., 2022). In this systematic review, 11 of 18 studies found that chronomodulated chemotherapy significantly decreased toxicity while maintaining efficacy. More specifically, chronomodulated chemotherapy reduced side effects including nausea, vomiting, mucositis and leukopenia for nasopharyngeal carcinoma ( Gou et al., 2018; Zhang et al., 2018), breast ( Coudert et al., 2008), colorectal ( Lévi et al., 1994, 1997) and endometrial cancer ( Gallion et al., 2003). Reducing side effects is a critical aspect of patient care as it improves quality of life and often allows for higher or more frequent doses. Although chronomodulated chemotherapy does appear to reduce side effects, it remains elusive whether chronotherapy improves drug efficacy or prognosis. For example, only three studies report higher response rate and longer survival in chronotherapy treated groups ( Gou et al., 2018; Lévi et al., 1994, 1997). However, two of the three of these studies report higher dose intensity in the chronotherapy treated group because of the reduced side effects. Therefore, it is unclear whether improved response is due to increased dose or a direct result of chronotherapy. A meta-analysis on factors impacting drug timing effects found that study size and whether or not the study was publicly registered as a clinical trial affected the reported efficacy of chronomedicine ( Ruben et al., 2021), suggesting that further studies are needed. In summary, optimal timing of anticancer drugs appears to reduce toxicity but more mechanistic studies are needed to determine the clinical relevance of anticancer chronotherapeutic approaches on drug efficacy.