The role of cryptochrome (CRY) in cancer: molecular mechanisms and clock-based therapeutic strategies

Abstract

The circadian rhythm is a phenomenon in which physiological, behavioral, and biochemical processes within an organism naturally fluctuate over a period of approximately 24 hours. This phenomenon is ubiquitous in living organisms. Disruption of circadian rhythms in mammals leads to different diseases, such as cancer, and neurodegenerative and metabolic disorders. In specific tissues, numerous genes have been found to have circadian oscillations, suggesting a broad role for rhythm genes in the regulation of gene expression. This review systematically summarizes the role of cryptochromes (CRYs) in the initiation and progression of different types of cancer and discusses the relationships between clock genes and the tumor microenvironment (TME), as well as clock-based therapeutic strategies.

Introduction

Organisms typically experience regular cycles of light and darkness, with the alignment of behavior to this circadian rhythm being advantageous for the prevalence of circadian phenomena. The molecular mechanisms of the circadian clock involve the production of Transcription-translation feedback loops (TTFLs), which interact with a set of CLOCK proteins to regulate the expression of specific target genes and their products in a circadian pattern. The core TTFL is modulated by four CLOCK proteins, including two activators, the circadian locomotor output cycles kaput (CLOCK) and brain-muscle binding protein 1 (BMAL1), and two inhibitors, period circadian protein homolog (PER) and CRY. The PER and CRY proteins combine to form a heterodimer in the cytoplasm, which then moves to the nucleus to hinder CLOCK/BMAL1 transcriptional activation. Following the degradation of the PER/CRY complex through a ubiquitin-dependent pathway, CLOCK/BMAL1 is liberated, initiating a new cycle. This 24-hour process establishes a fundamental circadian rhythm within the body ( Figure 1) [1]. Numerous genes have been shown to exhibit circadian oscillations in tissues, indicating the significant role of circadian genes in gene expression regulation. Disruptions in mammalian circadian rhythms have been linked to various diseases, such as cancer, neurodegenerative disorders, and metabolic conditions [ 2, 3].

Figure 1 .

Mammalian transcription-translation feedback loops (TTFLs)

PER, period circadian protein homolog; CRY, cryptochrome; RORs, RAR-related orphan receptors; REV-ERB, reverse orientation c-erbA-related receptor; CLOCK, circadian locomotor output cycles kaput; BMAL1, aryl hydrocarbon receptor nuclear translocator-like protein 1 (encoded by ARNTL); CCGs, clock-controlled genes; CK1, casein kinase 1; AMPK, AMP-activated protein kinase.

This review examines the impact of CRYs on the onset and progression of cancer and explores the associations between clock genes and the tumor microenvironment (TME), along with clock-based therapeutic strategies.

Physiological Function of the CRY Family

As early as 1993, the first cryptochrome gene, CRY1, was discovered in Arabidopsis and is related to the sensing of blue light and ultraviolet light. Cryptochrome is also found in other species, including insects, fish, amphibians, and mammals [4]. Cryptochromes are widely found in bacteria and eukaryotes but not in archaea. In mammals, the cryptochrome is an important component of the circadian clock and plays a key role in maintaining the circadian rhythm.

The general structure of the CRY protein is similar to that of DNA photolyase, but CRY is devoid of photolyase activity. In addition to the CRY-DASH protein, CRY is composed of two domains, an amino-terminal (PHR) region homologous to DNA photolyase and a carboxy-terminal domain of different sizes [4]. The PHR region binds to two chromatophores, flavin adenine dinucleotide (FAD) and 5,10-methylenetetrahydrofolate (pterin or MTHF), and is a cofactor for light absorption. However, the carboxyl terminus is more conserved.

The CRY family comprises CRY1 and CRY2, which play complex roles in different carcinomas. The CRY1 gene is located on chromosome 12q23.3, whereas CRY2 is located on chromosome 11p11.2. CRY1/2 is the main component of the negative feedback loop of circadian clock genes. These processes maintain normal cellular homeostasis pathway feedback, organic dynamic balance, and natural cell division. Moreover, CRY2 controls DNA damage checkpoints and regulates the expression of essential cell cycle genes by regulating the circadian clock gene network [5].

The Role of CRYs in Cancer

CRY proteins function differently in the malignant transformation and development of diverse tumor types ( Figure 2).

Figure 2 .

Proposed regulatory mechanism of CRY genes in cancer

cAMP, cyclic adenosine monophosphate; Pck1, phosphoenolpyruvate carboxykinase 1; AKT, serine/threonine-protein kinase; MMP2, matrix metalloproteinase-2; BRP, the key presynaptic protein bruochrome; Rb, retinoblastoma-associated protein; PI3K, phosphatidylinositol 3-kinase; EGFR, epidermal growth factor receptor; PKA, cAMP-dependent protein kinase; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6.

CRYs and liver cancer

Primary liver cancer ranks as the sixth most prevalent cancer globally and is the third leading cause of cancer-related mortality [6]. Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer, accounting for 90% of cases, whereas cholangiocarcinoma (CCA) accounts for 10%–15% of cases [7]. Risk factors associated with HCC include not only viral infections such as hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholism, and metabolic disorders but also nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), obesity, and diabetes [ 8, 9]. Consequently, numerous factors influencing lipid metabolism play crucial roles in the initiation and progression of HCC.

In the liver, approximately 10% of genes are rhythmically expressed [ 10, 11]. Metabolism in the liver also shows some rhythmicity. In 1981, North et al. [12] examined 13 metabolic enzymes in the mouse liver. These metabolic enzymes clearly have a circadian correlation. Circadian clock disruption accelerates the development of liver diseases such as fatty liver, cirrhosis, hepatitis, and liver cancer. The rhythmicity of clock gene expression is temporally shifted in the livers of tumor-bearing mice [13]. Recent studies have shown that the expression of CRY1 in cancer tissues and adjacent tissues is significantly lower than that observed in normal liver tissues and is related to tumor invasion and lymph node metastasis. Circadian rhythm disturbance in mice enhances diethylnitrosamine (DEN)-induced HCC through the upregulation of c-Myc and downregulation of p53, leading to cell cycle activation. In addition, circadian clock disorders cause overall changes in liver metabolism in mice, which not only promote the synthesis and storage of fat by accelerating cytoplasmic glycolysis but also increase oxidative stress and the synthesis of precursors that support rapid cell division, thus promoting liver fibrosis and primary liver cancer progression [14]. The findings of this study indicated that mice lacking the CRY1/ 2 genes demonstrate increased weight gain and disrupted glucose metabolism, along with elevated levels of insulin and resistance to insulin in the liver and muscle [15]. This could be attributed to the role of CRY proteins in modulating the function of cAMP response element-binding protein (CREB) in the liver, thereby restricting the process of fasting gluconeogenesis. The overexpression of CRY1 has been reported to reduce blood glucose levels and improve insulin sensitivity in obese mice with insulin resistance, which confirms that CRY1 can have antidiabetic effects on the liver [ 16, 17]. The CRY proteins can also impact the maintenance of glucose levels in the body by periodically suppressing the activity of the genes of glucocorticoid receptor and phosphoenolpyruvate carboxykinase 1 ( Pck1). Pck1 plays a significant role in regulating gluconeogenesis [18]. In summary, the CRY family may inhibit the development of HCC, but the specific molecular mechanisms involved are still under investigation.

CRYs and colorectal cancer

Colorectal cancer (CRC) is the third most commonly diagnosed cancer globally but ranks second in terms of mortality rates [6]. Various physiological processes within the gastrointestinal tract, including the peristaltic movement of intestinal segments, the enzymatic activity of the mucosa, the expression of transporters in the small intestine mucosa, and the proliferation rates of different cell types, all demonstrate discernible circadian rhythms [ 19– 22]. Research has revealed a correlation between disruptions in the circadian clock and the onset and progression of colorectal cancer [23].

The expression of CRYs is lower in colorectal cancer tissues than in normal tissues [24]. Low CRY1 expression is associated with age and gender and mRNA levels are even lower in older age groups and females. CRY2 is related to the location of CRC, and the level of CRY2 is lower in the transverse colon; however, no significant correlation was found between age and gender. At the molecular level, the upregulation of CRYs in colorectal cancer cells promotes proliferation and migration, whereas the downregulation of CRYs significantly reduces colony formation and metastasis.

F-box and WD repeat domain-containing 7 (FBXW7 or FBW7) is a component of a conserved complex of SKP1, CUL1, and F-box protein (SCF)-type ubiquitin ligases. FBXW7 expression leads to the degradation of CRY2 by increasing CRY2 ubiquitination and accelerating the turnover rate of CRY2. High expression of FBXW7 can downregulate CRY2 expression and increase the sensitivity of colorectal cancer cells to chemotherapy [25]. CRYs also mediate a variety of factors that affect the occurrence and development of colorectal cancer.

Wee1 is a key regulator of the cell cycle and a member of the serine/threonine protein kinase family. Compared with that in normal intestinal tissues, the expression of Wee1 in colorectal cancer cells is significantly greater [26]. Wee1 blocks cell division by inhibiting the G2/M phase transition through its action on CDK1 [ 27, 28]. In Cry1- and Cry2-deficient mice, Wee1 expression is increased in multiple tissues, and cell proliferation is inhibited [29]. These findings suggest that Wee1 expression is regulated by CRYs and affects the progression and development of CRC, but the specific mechanism remains unclear.

In the absence of CRYs, the expression of p53-related p73 gene is increased, thereby inducing the apoptosis of cancer cells in response to DNA damage [30]. The cAMP/PKA/RhoA pathway might mediate the tumor-promoting effect of CRY1 in CRC. CRY1 interacts with adenylyl cyclase, and an increase in CRY1 expression leads to a decrease in cAMP level upon exposure to prostaglandin E2 (PGE2) and isoproterenol [31]. PKA, a protein kinase associated with the cAMP signaling pathway, can inhibit the activity of Rho A [32]. Rho A plays a crucial role as a significant constituent of the Rho family of small GTP-binding proteins, facilitating the transmission of signals associated with the organization of the cytoskeleton, cellular proliferation, and migration. Reduced Rho A activity impedes tumor invasion and metastasis [33]. These results suggest that CRYs can promote the occurrence and development of CRC by affecting cell proliferation, cell migration, and DNA damage response.

CRYs and breast cancer

Breast cancer (BC) is a common malignant tumor in women. The incidence of breast cancer ranks first worldwide, and the mortality rate is fifth [6]. The incidence of BC is increasing at a rate of 3.1% per year [34]. In addition to well-established risk factors, such as menstruation, family history, and diet, circadian disturbance of the biological clock also contributes to the etiology of BC associated with changes in the social work environment [35]. For example, in night shift workers, the incidence of breast cancer is greater, which may be related to industrial production caused by the use of electric lighting at night causing the circadian rhythm to be disrupted [36].

CRYs function as tumor suppressors in BC. The expression of CRY2 is significantly lower in BC tissues than in normal tissues and is positively correlated with the degree of differentiation. In addition, the hypermethylation level of the CRY2 gene promoter is increased in BC tissues. Three SNPs (rs11038689, rs7123390, and rs1401417) of CRY2 are closely related to the risk of postmenopausal breast cancer. However, this association is only observed in women with estrogen receptor (ER)- and progesterone receptor (PR)-negative breast cancer but not in those with ER/PR-positive breast cancer [ 37, 38]. CRYs have been reported to be involved in the regulation of cell cycle progression and cell proliferation [5]. The CCND1 gene, which encodes cyclin D1, is often overexpressed in primary BC. Studies have shown that CRY2 knockdown induces the overexpression of cyclin D1, thereby accumulating DNA damage and disrupting cell cycle progression, thus promoting the development of BC [39]. Moreover, an epidemiological study revealed that TIMELESS is associated with the risk of BC [40]. TIMELESS is known as a positive regulator of DNA replication, and its high expression is related to the proliferation of set genes and is associated with poor prognosis. Recently, TIMELESS was reported to physically interact with the core clock element CRY1, and PER2 competes with TIMELESS to bind CRY1. The high expression of TIMELESS and the low expression of PER2 in cancer cells lead to a decrease in the PER2-CRY1 dimer, which is beneficial to the TIMELESS-CRY1 dimer. These actions can antagonize the ability of the PER-CRY heterodimer to regulate the expression of c-Myc and cyclins D1, B1, and A2. This might also explain the coexistence of high TIMELESS expression, low levels of other core clock components, and high proliferation of more aggressive BCs [41]. In breast cancer with insulin resistance, the transcription factor forkhead box protein K1 (FOXK1) undergoes posttranslational modifications mediated by O-GlcNAc transferase (OGT) and is translocated to the nucleus. Consequently, it binds to multiple corepressor complexes, including the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and SIN3A, leading to the suppression of CRY2 transcription. This, in turn, disrupts the circadian rhythm and promotes the development of breast cancer [42]. Thus, CRYs might be used as biomarkers for estimating the susceptibility, progression, and prognosis of BC.

CRYs and osteosarcoma

Osteosarcoma (OS) is the predominant type of primary malignant bone tumor that arises from undifferentiated mesenchymal cells and is responsible for bone formation. The age of onset of OS exhibits a dual peak distribution, with the initial peak occurring during adolescence and the subsequent peak in adulthood. The precise etiology of OS remains ambiguous. Established risk factors linked to the onset of OS include exposure to ionizing radiation, alkylating agents, Paget’s disease, hereditary retinoblastoma, Li-Fraumeni familial cancer syndrome, and various chromosomal abnormalities [43].

CRY family members might also act as tumor suppressors in OS. Compared with that in normal tissues, a reduced expression level of CRY1 in human OS tissues has been observed. CRY1 silencing leads to the activation of the Akt/p53/p21 pathway. The phosphorylation level of Akt increases with the downregulation of CRY1, which activates MDM2 to inhibit the p53/p21 cascade, thus promoting cell cycle progression to facilitate the proliferation of OS cells [44]. CRY2 has similar effects on OS. The downregulation of CRY2 results in elevated levels of matrix metalloproteinase 2 (MMP2) and β-catenin, leading to enhanced proliferation and migration of OS cells through the activation of the mitogen-activated protein kinase (MAPK) and Wnt/β-catenin signaling pathways [45]. STAT3 can transactivate miR-7-5p, which inhibits the binding of CRY2 to the CLOCK/BMAL1 dimer, thereby facilitating CLOCK/BMAL1 binding to the p300 E-box promoter region to stimulate its transcription. Subsequently, p300 promotes histone 3 acetylation and forms a transcriptional complex with Runx2, thus enhancing osteogenesis [46]. Accordingly, CRYs might be potential biomarkers for the diagnosis of OS, and CRY agonists could be developed as therapeutics against this disease.

CRYs and glioblastoma

Malignant brain cancer is one of the most feared tumors, not only because of its high mutation rate and poor prognosis but also because it has a serious impact on the quality of life and cognitive function of patients. Glioblastoma (GBM) and malignant glioma are the most common primary malignant brain tumors. Unlike in many other types of cancer, CRY family members might act as oncogenes to promote the occurrence and development of GBM. GBM can be divided into two circadian clock-related subtypes, CC1 and CC2. In the CC1 subtype, CRY2 is overexpressed, whereas CRY1 is overexpressed in the CC2 subtype [47]. The expression level of CRY1 is negatively correlated with overall survival and is associated with poor prognosis in patients with GBM [48]. The mechanism of GBM inhibition induced by CRY downregulation might involve its interaction with BRUCHPILOT (BRP), which is a key presynaptic protein regulating the synaptic number. In fruit flies, mutation of CRYs leads to decreased BRP expression. Therefore, the overexpression of CRYs in glial cells might promote the establishment of synapses with neurons. Loss of light input impairs the degradation of BRP in glial cells and promotes tumor progression [49]. Typical signal transduction and tumor suppressor pathways in GBM include the p53, Rb, and PI3K pathways [50]. Myc resides at the intersection of the EGFR and PI3K pathways and is extensively involved in the regulation of cell proliferation, transcription, differentiation, apoptosis, and cell migration. Approximately 60%–80% of human GBM cases exhibit elevated Myc expression [51]. Glial cells have also been reported to transform into gliomas through the activation of the PI3K and EGFR pathways. CRY2 serves as a crucial cofactor in the ubiquitination of Myc mediated by SCFFBXL3 [52]. The histone demethylase KDM4B regulates the stability of MYC through the SCFFBXL3-CRY2 E3 ligase complex, accelerating the progression of GBM [53]. CRYs mediate the accumulation of Myc in glioma cells by upregulating the expression of these signaling pathways [54].

CRYs and leukemia

The process of hematopoiesis is tightly controlled and involves the differentiation and maturation of stem cells into various cell types, such as red blood cells, megakaryocytes, and immune cells of myeloid, lymphoid, or monocytic lineages within the bone marrow or lymphoid tissue. In this process, various abnormal factors can hinder the differentiation and maturation of hematopoietic stem cells at different stages, leading to the proliferation of immature leukemic immune cells, i. e., leukemia. Studies have shown that circadian rhythm disorders contribute to the tumorigenesis and development of leukemia [ 55, 56].

In the absence of CRYs, the inhibition of adenylate cyclase (AC) is relieved, which leads to increased cAMP production and PKA activation. These actions increase the phosphorylation level of p65 and ultimately result in the expression of downstream target genes such as p21 and proinflammatory cytokines, such as TNF-α and IL-6 [ 31, 57]. The expression level of TNF-α is positively correlated with the clinical endpoint of leukemia [ 58, 59]. Interleukin-6 (IL-6) is the most typical cytokine associated with inflammation and plays an important role in the regulation of hematopoiesis and the formation of leukemia cells. In CRY1- and CRY2-knockout mice, the expression of IL-6 and TNF-α is significantly increased [31]. The lack of CRYs activates proinflammatory cytokines, suggesting a potential role for CRY proteins in regulating inflammatory cytokine expression. Therefore, CRYs may affect the occurrence and development of leukemia cells through different pathways, but their diagnostic value as biomarkers of leukemia needs to be confirmed by further studies.

CRYs and other cancer types

In addition to the cancer types mentioned above, CRYs have also been occasionally reported in other malignancies. In lung adenocarcinoma, a lower expression level of CRY2 has been observed than in paracancerous tissues, and higher expression of CRY1 and CRY2 correlates with better overall survival of patients. Therefore, CRYs can be used as new therapeutic targets and prognostic biomarkers for lung adenocarcinoma [60]. In the DEN-induced HCC model, the incidence of cholangiocarcinoma is significantly increased in CRY1/ 2 double-knockout mice. This increase might be caused by bile acid accumulation and cholestasis in Cry double-deficient mice; however, the specific mechanism remains to be explored [61]. Additionally, CRY1 expression levels are lower in melanoma tissues than in adjacent nontumor tissues [62]. With deeper exploration of the function and mechanism of the CRY family in various diseases, targeting CRY and its regulatory network might be developed as a new therapeutic strategy for numerous diseases.

Take together, the function and mechanism of CRYs in various cancers are summarized in Table 1.

Table 1 The function and mechanism of CRYs in cancer

Name	Cancer type	Cancer suppressors/promoters	Regulatory mechanism	Effect of CRY genes on cancer progression	Ref.
CRY1	Liver cancer	Cancer suppressors	cAMP↓creb phosphorylation↓	Reduces fasting gluconeogenic gene expression.	[18]
Colorectal cancer	Cancer promoters	p73↓	Enhances sensitivity to DNA damage-induced apoptosis.	[63]
Wee1↓	Promotes proliferation.	[29]
cAMP/PKA/RhoA↓	Promotes tumor invasion and metastasis.	[ 31– 33]
Breast cancer	Cancer suppressors	TIMELESS-CRY1 dipolymer↑PER2-CRY1 dipolymer↓	Regulates the expressions of c-Myc and cyclins.	[41]
Osteosarcoma	Cancer suppressors	Akt/P53/p21↓	Inhibition of proliferation.	[44]
Glioblastoma	Cancer promoters	BRP↑	Promote the establishment of synapses with neurons.	[49]
Leukemia	Cancer suppressors	cAMP/PKA↓	Restricts the expression of pro-inflammatory cytokines (TNF-α, IL-6).	[ 31, 57]
Gastric cancer	Cancer promoters	cAMP↓ERK1/2 phosphorylation↑	Protects cancer cells from the antiproliferative effects of ISO.	[64]
CRY2	Liver cancer	Cancer suppressors	Regulates triglyceride synthesis a	Promotes the storage of triglycerides and limit the production of glucose.	[65]
Colorectal cancer	Cancer promoters	p73↓	Enhances sensitivity to DNA damage-induced apoptosis.	[63]
Wee1↓	Promotes proliferation.	[29]
Cancer suppressors	Promotes c-MYC ubiquitination b	Inhibition of proliferation.	[66]
Breast cancer	Cancer suppressors	CD44↓Methylation of BCL2↑	Inhibition of invasion.	[67]
Osteosarcoma	Cancer suppressors	MAPK↓Wnt/β-catenin↓	Inhibition of proliferation and migration.	[45]
Glioblastoma	Cancer promoters	p53/c-Myc/Rb/PI3K/EGFR↑	Promotes the stability of c-Myc.	[ 50– 54]
Leukemia	Cancer suppressors	cAMP/PKA↓	Restricts the expression of pro-inflammatory cytokines (TNF-α, IL-6).	[ 31, 57]

^aThe specific mechanism is unknown. ^bIn collaboration with FBXL3. cAMP, cyclic adenosine monophosphate; creb, cyclic AMP-responsive element-binding protein 1; PKA, cAMP-dependent protein kinase; RhoA, transforming protein RhoA; AKT, serine/threonine-protein kinase; BRP, BRUCHPILOT; PI3K, phosphatidylinositol 3-kinase; EGFR, epidermal growth factor receptor; ERK1/2, extracellular-signal-regulated kinase; FBXL3, F-box/LRR-repeat protein 3; Bcl-2, apoptosis regulator Bcl-2.

Circadian Rhythm and Tumor Microenvironment (TME)

Tumor cells do not exist in isolation. The internal environment in which tumor cells arise and reside is called the TME. Typically, the TME consists of an extracellular matrix (ECM) and a variety of cell subsets. These include innate myeloid cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and dendritic cells. The TME also includes lymphocytes, such as T cells and natural killer (NK) cells. Cancer-associated fibroblasts (CAFs) and endothelial cells are also found in the TME [68]. In recent years, the role of the TME in tumor progression and treatment efficacy has been widely reported. The TME is regulated by many factors, among which the circadian clock has gradually become a focus of research. Therefore, exploring the regulation of the circadian clock associated with the TME will help in the development of new treatment strategies.

As mentioned earlier, clock genes have important effects on the occurrence and development of cancer cells. The clock genes can also affect the TME and the interaction between cancer cells and the TME [69]. For example, in an experimental setting where colon cancer cells are cultured alongside normal fibroblasts obtained from human colorectal tumor samples, colon cancer cells exhibit changes in their circadian rhythm and metabolic characteristics. Additionally, they display reduced apoptosis, enhanced viability, and increased resistance to chemotherapeutic drugs. The interaction between colon cancer cells and tumor-associated fibroblasts influences the molecular clock mechanism and promotes the development of colorectal cancer [70]. Chronic circadian perturbation was performed in a mouse model of spontaneous breast tumorigenesis. In this study, circadian disruption significantly increased cancer cell dissemination and lung metastasis. It also enhances the stemness and tumor-initiating potential of tumor cells and creates an immunosuppressive shift in the TME [71]. In a mouse melanoma model, circadian rhythm disruption not only led to faster tumor cell growth and shorter latency but also caused an imbalance between M1 (proinflammatory) and M2 (anti-inflammatory) macrophages in the spleen and tumor tissue, inducing immune tolerance and promoting tumor growth [72]. Thus, the circadian rhythm can regulate the TME and subsequently affect the occurrence and development of tumors.

Clock-based Therapeutic Strategies

Clock-based therapeutic strategies refer to a treatment plan on the basis of the characteristics of the human circadian clock. Biological rhythm affects the metabolism and curative effect of antitumor drugs. Thus, following the biological rhythm to explore the best dosing regimen might improve therapeutic outcomes [73].

There are mainly three Clock-based therapeutic strategies. The first is to optimize lifestyle by maintaining the circadian rhythm of the body. Exogenous melatonin is indeed effective at improving sleep latency but has little effect on maintaining sleep [74]. Thus, improving sleep quality by training for regular circadian rhythms might indirectly improve other aspects controlled by the circadian clock, thereby invigorating physical health.

The second strategy is to optimize therapy timing, also known as timing administration. Studies have shown that the mitotic index of human cancer cells varies greatly among individuals. Therefore, choosing the appropriate chemotherapeutic time is critical. In other words, targeting the peak of DNA synthesis in cancer cells, rather than normal cells, might maximize the cytotoxic drug effect on cancer cells while protecting normal cells and reducing side effects. In addition, drug efficacy, as well as toxicity, may change over time. Cisplatin, a first-line antitumor drug, has been demonstrated to exhibit certain circadian rhythm-dependent sensitivity [75]. The pharmacokinetics of drugs, as well as their metabolism in the human body, may change with circadian rhythms. Therefore, finding the appropriate time for the administration of anticancer drugs is necessary to improve the therapeutic effect and reduce side effects [76].

The last strategy is to target specific biological clock components, which have emerged as novel therapeutics for chronic diseases and cancer. A series of interacting factors have been reported to affect the stability of CRYs through posttranslational modifications. FBXL3 is an F-box-type ubiquitin E3 ligase that promotes the ubiquitination and degradation of CRYs [ 77, 78]. FBXL21, the closest homolog of FBXL3, ubiquitinates CRYs but leads to the stabilization of CRYs [79]. Ubiquitin-specific protease 7 (USP7, also known as HAUSP) binds to both CRY1 and CRY2 and stabilizes CRY1 via deubiquitination. Treatment with USP7-specific inhibitors or Usp7 knockdown shortens the circadian cycle of the cell rhythm. TAR DNA-binding protein 43 (TDP-43) stabilizes CRY1 and CRY2, and its knockdown also shortens the circadian rhythm of cultured cells [80]. PER, CRY2, and CK1 can form ternary complexes. PER2 and CRY2 are phosphorylated by CK1 and may then be degraded through the ubiquitin‒proteasome pathway [81]. Ser557 in the C-terminal tail of CRY2 is phosphorylated by dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A), which acts as the initiating kinase for subsequent glycogen synthase kinase 3β (GSK-3β)-mediated phosphorylation of Ser553, leading to proteasomal degradation of CRY2. However, CRY1 protein levels are not affected by DYRK1 [82]. CRY2 can be driven by p300 to undergo acetylation, thereby stabilizing CRY2. However, histone deacetylase 6 (HDAC6) can reverse this process [83]. The CRY stabilizer KL001 inhibits FBXL3-mediated ubiquitination of CRY1/2 by competitively occupying the folacin adenine dinucleotide (FAD) binding pocket, thereby directly interacting with CRY1/2 and stabilizing their protein levels. KL001 treatment inhibits glucose synthesis in hepatocytes and is causally expected to be applied as a therapeutic against metabolic disorders [84]. KS15 is an inhibitor of CRYs, and its addition hinders CRY1/2-mediated binding and transcriptional inhibition, thereby activating CLOCK-BMAL1-E-box-mediated transcription [85]. Thus, KS15 promotes the expression of tumor suppressor genes and enhances the sensitivity of cancer cells to chemotherapy.

On the basis of the above three important approaches ( Table 2), the circadian rhythm is obviously inseparable from disease, and clock-based treatment strategies can play a crucial role in optimizing treatment effects and have excellent prospects for clinical application.

Table 2 Summary of clock-based therapeutic strategies

Strategy	Theory	Drugs	Ref.
Leading a regular life	Maintaining a regular lifestyle and optimizing the lifestyle to keep the body healthy.	Melatonin	[74]
Timing administration	Targeting the peak of DNA synthesis in cancer cells, rather than normal cells, might maximize the cytotoxic drug effect on cancer cells while protecting normal cells and reducing side effects. The metabolism of drugs in the human body may change with the circadian rhythm.	Cisplatin, Taxol, 5-Fu, Capecitabine	[ 86– 88]
Targeting CRY or other CRY-interacting molecules	Rhythm disorder is one of the important causes of cancer. By targeting abnormal clock components in cancer cells, the therapeutic effect can be improved.	KL001, KS15	[ 84, 89]

Summary

Circadian rhythm disruption is a significant factor contributing to the development of tumors. The malfunction of CRYs and other genes within the clock gene family plays a role in this phenomenon. The regulation of DNA replication and cell cycle progression in healthy cells is closely controlled by various cell cycle regulators. Notably, several key cell cycle regulators, including CDK6 (G1/S), Cdc25b (G1/S, G2/M), p21, p57, and certain cyclins, exhibit daily rhythmic expression patterns [90]. Furthermore, tumor suppressor genes and oncogenes also exhibit circadian rhythm-dependent expression. For example, the p53 protein, a crucial tumor suppressor, undergoes circadian oscillations in its transcription, stability, and activity. Research indicates that mutations in the CRY2 gene (e.g., D325H and S510L) promote the proliferation of primary mouse fibroblasts with elevated c-Myc levels while also suppressing the expression of p53 target genes [91].

A close correlation is observed between DNA damage response (DDR) pathways, DNA repair, and circadian rhythm mechanisms. The expression of many DNA repair genes has a circadian rhythm [90]. CRYs are structurally similar to photo repair enzymes involved in DNA single-strand break repair. Although mammalian CRYs lack DNA repair activity, CRY1 promotes the interaction between ATR and TIMELESS, resulting in the circadian rhythm of ATR activity [92]. Similarly, CRY2 may promote the interactions among ATR, TIMELESS, and Chk1 [93].

The replication potential of normal cells is limited, whereas cancer cells are immortalized, which may be related to the expression of telomerase at the ends of chromosomes. The maintaining of the length of chromosome ends or telomeres by telomerase is also controlled by the biological clock. The expression of telomerase can be directly regulated by BMAL1/CLOCK through the E-box in the telomerase reverse transcriptase ( TERT) gene promoter; thus, TERT displays circadian rhythmic expression. Accordingly, the clock genes might promote the immortalization of cancer cells by affecting the expression of TERT.

Compared with healthy cells, cancer cells present distinct metabolic needs, potentially stemming from disruptions in the regulation of the circadian clock. The increased glycolytic activity observed in cancer cells, known as the Warburg effect, serves to fuel these cells with energy. Multiple pathways are implicated in the upregulation of glycolysis in cancer cells, with constitutive activation of the PI3K/Pdk1/Akt pathway being identified as a key driver of glycolytic metabolism in cancer cells [94]. Moreover, hypoxia in tumors activates hypoxia-inducible factor 1 (HIF1), thereby increasing the transcription of target genes, such as glucose transporter 1 ( GLUT1) and lactate dehydrogenase A ( LDHA). HIF1 has been shown to interact with PER2 to regulate the metabolic balance of cardiomyocytes under hypoxic and ischemic conditions [95]. These genes are involved in glucose transport and metabolism in glycolysis [96]. The protein accumulation of components of the Akt pathway displays circadian rhythms, and the activation of this signaling also affects the circadian rhythm.

The key elements of the immune system exhibit circadian rhythms and maintain homeostasis during instances of inflammation. Disruptions in circadian rhythms can affect various immune system functions and contribute to persistent inflammation. Research has demonstrated that certain fundamental clock genes in mice can impede the production of numerous proinflammatory cytokines in cases of chronic inflammation. Manipulation of the genetic makeup of circadian clock components, such as BMALL and CRYs, has been found to influence the levels of multiple cytokines and chemokines, including GM-CSF, CCL2, CXCL6, CCR2, CX3CR1, IL-2, IL-10, IL-6, IL-19, IL-1β, TNF-α, MCP-1/JE, MMP9, and IFN-γ. In pancreatic cancer cells, the transcription of CRY1 is dependent on SMAD family member 4 (SMAD4). The activation of TGF-β leads to circadian rhythm disorders and increases the invasiveness of cancer cells [97]. An abnormal circadian clock also results in T cell dysfunction and immune escape of cancer cells via the upregulation of immune checkpoint molecules, such as PD-L1 and CTLA-4 [98].

These possible mechanisms closely link CRYs, as well as other clock gene family members, with the carcinogenesis and progression of tumors. They also provide strong evidence for the subsequent targeted treatment of malignant tumors on the basis of the circadian rhythm principle. Considering the differential expression of CRY genes in different tumors, these targeted therapies may also provide new therapeutic strategies. Moreover, rhythm genes involved in the cell cycle, DNA damage, and cell metabolism exhibit rhythmic expression. Circadian rhythms also influence the TME and the interaction between cancer cells and the TME. A deep understanding of the mechanisms by which clock genes regulate these physiological and pathological processes will be helpful in identifying important biomarkers to predict cancer risk and chemotherapy sensitivity in the future.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grant from the National Natural Science Foundation of China (No. 82173144).