Major roles of the circadian clock in cancer

Circadian rhythms are natural rhythms that widely exist in all creatures, and regulate the processes and physiological functions of various biochemical reactions. The circadian clock is critical for cancer occurrence and progression. Its function is regulated by metabolic activities, and the expression and transcription of various genes. This review summarizes the composition of the circadian clock; the biological basis for its function; its relationship with, and mechanisms in, cancer; its various functions in different cancers; the effects of anti-tumor treatment; and potential therapeutic targets. Research in this area is expected to advance understanding of circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like protein 1 (BMAL1) in tumor diseases, and contribute to the development of new anti-tumor treatment strategies.


Introduction
The circadian clock regulates various physiological processes and biochemical reactions in the human body. This system exhibits diurnal variation characteristics with time, which are denoted circadian rhythms 1 . Circadian rhythms are relatively conserved in evolution. They are widespread in various organisms and have evolved 4 times with selective advantages 2 . Because of its rhythm, the system is also referred to as the circadian clock system. This system comprises the central and peripheral clocks located in the hypothalamus's anterior suprachiasmatic nucleus (SCN). The central and peripheral clocks have a synchronous circadian rhythm 3,4 . The central clock's function can be performed independently, whereas the peripheral clock is coordinated through various signaling molecules to achieve synchronization.
Transcription-translation feedback loop (TTFL) is the molecular basis of circadian rhythms in organisms. TTFL regulates the central molecular circadian clock mechanism 1,5-7 . TTFL often plays a role in the SCN and peripheral tissues of the anterior hypothalamus of mammals. Optical signals are the main factors affecting the circadian clock. The optical signal stimulation received by optic nerve fibers generates downstream nerve or endocrine signal stimulation through the SCN, thereby synchronizing with peripheral organs [6][7][8][9][10][11] .
The positive stimulus factor TTFL comprises circadian locomotor output cycles kaput (CLOCK), aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL), and its paralog NPAS2. ARNTL is also called brain and muscle ARNTlike protein 1 (BMAL1). TTFL binds the target sequence E-box (CTGCAG), thereby promoting the expression of transcription inhibitor cryptochrome (CRY1/2) and period (PER1/2/3), whereas CRY and PER serve as negative stimuli for TTFL [12][13][14] . Two different stimuli can form 2 complexes with opposite functions: the CLOCK-BMAL1 transcription activator and CRY-PER transcription inhibitor. Their growth and decline exhibits a clear circadian rhythm. As illustrated in Figure 1, the CRY-PER complex enters the nucleus and inhibits the function of the CLOCK-BMAL1 complex.
The above feedback loop is also consolidated by a secondary feedback loop. In the secondary feedback loop, the CLOCK-BMAL1 complex acts on the nuclear receptor Rev-ERBα/β(NR1D1/2) and retinoic acid receptor-related orphan receptors (RORs), and regulates their expression levels, thereby affecting BMAL1 expression 1,5 . Additionally, this loop is controlled by the kinase CK1δ/ε and ubiquitin ligases [15][16][17][18][19][20] . However, this basic understanding of the molecular mechanism underlying circadian rhythm in mammals is insufficient for routine medical care. Additional information is required regarding the mechanisms.
At the beginning of the circadian clock, owing to the presence of the negative regulatory factor CRY [12][13][14] , the CLOCK-BMAL1 complex cannot activate transcription even if it binds the E-box sequence. However, after a light period (midday of the circadian clock), CRY separates from the CLOCK-BMAL1 complex. Subsequently, because of the loss of CRY inhibition, transcription at the E-box is initiated, thereby promoting the expression of CRY and PER. Although PER continually accumulates, it cannot inhibit E-box transcription in the absence of CRY mediation. In addition, the CLOCK-BMAL1 complex acts on the nuclear receptor Rev-ERBα/β (NR1D1/2) and RORs and regulates their expression levels, thereby affecting the expression of BMAL-1 1,5   the form of CRY-PER-CK1δ, and PER mediates the phosphorylation of CK1δ on CLOCK. The new CLOCK-BMAL1 complex with CRY replaces the complex that bound the E-box in the previous phase. This inhibition differs from that mediated by CRY. Finally, the CLOCK-BMAL1 complex combined with CRY binds the E-box before a new cycle begins 21 .
Several studies have revealed a close relationship between circadian rhythm disorders and cancer. A meta-analysis of various human cancer transcriptome datasets has revealed the dysregulated expression of circadian genes in different tumor types 22 . First, a mutual regulatory mechanism exists between circadian and cancer genes. The genes regulated by the circadian clock include various oncogenes and tumor suppressor genes, and the core genes of the circadian clock are also regulated by oncogenes and tumor suppressor genes, which are involved in tumor onset and malignancy 23,24 . Second, the circadian clock regulates gene rhythms associated with metabolic and endocrine functions, and metabolic activity and endocrine homeostasis play important roles in tumor development [25][26][27][28] . In addition, abnormal biological rhythms promote the malignant progression of tumors by weakening the body's immunity. Immunity is an important factor restricting tumor development. Disrupting biological rhythms not only affects the innate and acquired immunity of the body, but also promotes tumor immune escape through immune checkpoints [29][30][31][32] . In summary, the circadian clock may play an important role in the entire process of tumorigenesis, and has the potential to be applied in tumor prevention, diagnosis, and treatment.

Methods
In the analysis accompanying this review, RNA-seq data (level 3) and corresponding clinical information on pan-cancer were obtained from the Cancer Genome Atlas (TCGA) database (https://portal.gdc.com). For the predictive analysis of single genes in multiple tumors, univariate Cox regression analysis and a forest plot constructed with the "forest plot" R package revealed the P-values, HR, and 95% CI. Tumor mutation burden (TMB) was derived from The Immune Landscape of Cancer, published by Vesteinn Thorsson in 2018 33 . Immune-associated assessment was performed with xCell analysis. All bioinformatics analyses were performed in R software v4.0.3 for statistical analysis. If no additional explanation is provided, the rank-sum test was performed to detect the differences between the 2 groups of data, and P < 0.05 was considered statistically significant.

Circadian clock and cancer
Human genes are highly similar to mouse genes. Previous studies have found that nearly half the protein-coding gene expression in mice is associated with the circadian rhythm. The circadian clock controls the rhythmic expression of some genes in organisms under TTFL regulation, and these genes further regulate the expression of other genes. A total of 50%-80% of protein-coding genes in mice and humans have been found to show rhythmic oscillations 5 . The circadian clock is widely expressed in human genes and is influenced by environmental factors (light and food). When the circadian clock rhythm is perturbed by various factors, the normal biological characteristics of human organs or cells are altered. These changes may be correlated with the pathogenesis of various diseases, including some psychological diseases, endocrine diseases, and even cancer 7,10,34-39 . The physiological activities, cell metabolism, proliferation, and differentiation in the human body all show rhythmicity. Cancer cells, which abnormally proliferate in the human body, may be more susceptible to circadian clock disorders than normal cells because of their unstable characteristics.
Are circadian clock disorders associated with cancers? At present, the controversy regarding this issue has related primarily to several earlier epidemiological studies and recent studies on the clear correlation between clock disorders and cancers. Previous epidemiological investigations have revealed a lack of correlation between circadian clock disorders and the development of specific cancers. However, according to the International Agency for Research on Cancer (IARC) and other recent research, when the human circadian clock is disrupted, the likelihood of developing cancer, including lung cancer, intestinal cancer, and breast cancer, dramatically increases 5,40-46 . The above controversy may be due to the use of different research methods, or differences in sample size and representativeness. In clinical practice, the influence of circadian rhythms on human malignant tumors has been observed. Lou et al. 47 have investigated thyroid nodules in older people with varying degrees of malignancy, and concluded that poor sleep quality and biological rhythm disorders are independent risk factors. CLOCK and BMAL1 expression levels were found to be considerably higher in the malignant thyroid nodule group, whereas CRY2 expression was significantly lower. In addition, with the popularity of next-generation sequencing, bioinformatics technology has provided further evidence. The bioinformatics analysis depicted in Figure 2 supports a correlation between clock disorders and cancers. In different cancers, BMAL1 expression significantly differs between tumor and normal tissues. PER and CRY expression are detailed in the Supplementary Materials.
In addition to the above evidence, animal experiments have demonstrated a relationship between circadian rhythms and cancer. When CLOCK and BMAL1 mutations are artificially introduced, premature aging has been observed in mice that are not predisposed to cancer 48,49 . According to Papagiannakopoulos 50 , prolonged jet lag dramatically exacerbates cancer development and progression in K-Ras and p53 deficient mice.

The mechanisms of the circadian clock in cancer progression
We investigated the mechanisms underlying the effects of the circadian clock in cancer and assessed their similarities. The mechanisms may include the following: (1) Tumor cells undergo a variety of biochemical reactions, including cell growth and senescence, cell proliferation and apoptosis, DNA damage repair process, and various metabolic proces ses 1,5,36,44,[51][52][53][54][55] ; the circadian clock may affect tumor occurrence and development by regulating these reactions 44,56 . (2) Cancer stem cells (CSCs) undergo tumorigenesis, development, and metastasis, and the circadian clock plays a crucial role in the stemness of self-renewal cancer cell subsets, such as acute myeloid leukemia (AML) and pleomorphic glioblastoma In addition, immune escape is an integral part of cancer progression 63 . According to Chen et al. 58 , changes in CD8 + T cells often affect CLOCK expression in patients with glioblastoma multiforme. In the 4T1 mouse model of breast cancer, the CLOCK component has been found to induce regular expression of Wnt family member 10A (Wnt10A) and upregulate the expression of downstream acetaldehyde dehydrogenase 3 (ALDH3A1). ALDH3A1 is a characteristic of CSCs and is associated with the degree of tumor malignancy 66,67 . Moreover, previous studies have indicated that the depletion of T cells and up-regulation of programmed death-ligand (PD-1) in patients with cancer may be associated with the widespread mutation and genomic instability of the CLOCK gene 32,52 . This evidence suggests that CLOCK gene expression in cancer cells may contribute *** *** *** *** − *** *** *** to immune escape. Figure 3 shows these relationships more intuitively.

CLOCK and the potential molecular mechanisms of tumorigenesis Ghrelin
Ghrelin is a 28-amino acid peptide that acts a ligand for GHSR. It activates and promotes appetite, and affects many physiological activities, such as glucose metabolism and gastric acid secretion 68  the circadian rhythm [81][82][83] . After knockout of the BMAL1 gene in mice, the circadian rhythm fluctuation of Ghrelin disappears, thus suggesting a specific relationship between BMAL1 and Ghrelin; these rhythm-associated characteristics may be associated with tumorigenesis 84 .

Unfolded protein response (UPR)
The UPR is a stress response caused by protein accumulation in the endoplasmic reticulum, owing to abnormal protein folding into secondary structures. Additionally, it is a cell checkpoint pathway triggered by incorrect folding of the endoplasmic reticulum. The UPR involves Protein kinase RNA-like ER kinase (PERK), Activating Transcription Factor 6 (ATF6), and Inositol-requiring enzyme 1 (Ire1) 85 . miR-211 is a protein kinase RNA-like ER kinase-induced micro-RNA. According to Bu et al. 86 , miR-211 regulates BMAL1 expression and CLOCK composition. BMAL1 inhibition impairs UPR-dependent protein synthesis and affects the adaptation of cells to cell stress caused by endoplasmic reticulum homeostasis damage. Moreover, Bu et al. have discovered that the UPR inhibits BMAL1 in Burkitt lymphoma, thereby affecting circadian rhythm and protein synthesis, and promoting tumor progression, which may be associated with the continual activation of c-myc. Knockout of PERK in c-myc-driven lymphoma has been found to lead to ER protein overload and cancer cell death 87 . Inhibition of BMAL1 by PERK-miR-211 is crucial for promoting the progression of c-myc-positive lymphoma. These results demonstrate that PERK-induced miR-211 inhibits BMAL1 expression, limits protein overload, and promotes tumor progression.

Effects on anti-tumor immunity by regulation of TAM metabolism via Hif-1α
Hif-1α is a major factor regulating glycolysis and the synthesis of metabolic gene transcription and is also crucial for the synthesis of arginase-1 (Arg-1), a urea-cycling enzyme [88][89][90] . Previous research has indicated that tumor-associated macrophages (TAMs) upregulate Arg-1 through Hif1-α 84 , a macrophage marker. Anti-tumor immunity mediated by cytotoxic T-cell nuclear natural killer cells is an important factor affecting tumor progression. Arginine depletion in TAMs inhibits this process, which is associated with the abnormal expression of Arg-1 91,92 .
We previously discovered that fluctuations in circadian rhythm regulate the inflammatory function of macrophages. Knockout of BMAL1 increases mortality in septic mice. BMAL1 controls the rhythm of monocyte recruitment, thus playing a crucial role in the immune response to pathogens 93 . Alexander et al. 94 have demonstrated that macrophages induce BMAL1 expression after stimulation (inflammation, etc.), thereby aggravating Hif-1α-dependent metabolic reprogramming and causing mitochondrial dysfunction. The balance between BMAL1 and Hif-1α affects the metabolism of macrophages and anti-tumor immunity. BMAL1 gene deletion promotes tumor growth.

Myc
Oncogenes are genes that cause malignant transformation of cells. Dysfunction in the major oncogene family Myc or abnormal proliferation often leads to the occurrence and adverse prognosis of different tumors [95][96][97] . Myc and its partner MAX form E-box motifs similar to those of the BMAL1 gene that control circadian rhythms. Therefore, Myc also participates in cell growth, proliferation, and death 98,99 .
Previous studies have reported that Myc enhances the transcriptional activity of E-box sites and increases REV-ERBα/β expression, thus inhibiting BMAL1 function 100 . Myc expression is negatively correlated with the circadian clock 23 . As described above, PER is a negative stimulus of TTFL, and the CRY-PER complex inhibits the function of the CLOCK-BMAL1 complex. This inhibitory effect of PER has been proposed to be achieved through Myc [101][102][103] .
According to Anastasia et al. 104 , c-Myc plays a major role in CLOCK oscillators. Myc expression has a strict circadian rhythm, but its expression also responds to CLOCK function. Concurrently, PER and BMAL1 inhibit the transcription of c-Myc during tumorigenesis and development. Deletion of either of these 2 genes increases c-Myc expression, as demonstrated in a mouse lung cancer model 50 .

Ras Homolog Family Member A (RhoA)
The Rho family is involved in the occurrence and development of various tumors. Its GTPase activity regulates cell transformation, division, and angiogenesis 105 . RhoA regulates the adhesion, aggregation, and morphology of various cells by controlling actin and myosin contraction 106,107 . ROCK is the downstream effector of RhoA, and cofilin (CFL) is an actin-depolymerizing factor. Ma et al. 108 have demonstrated that CLOCK and BMAL1 regulate the dynamic conversion of F-/G-actin by controlling the RhoA-ROCK-CFL pathway, thereby promoting the proliferation and invasion of tumor cells, which may be associated with inhibition of CUL3-mediated ubiquitination and an increase in RhoA expression.

Protein kinase
Several kinases regulate Clock/Bmal and other circadian genes, such as AMP-activated protein kinase (AMPK), Akt, and mammalian rapamycin target protein (mTOR). Their roles are crucial for cancer development. Protein kinases involved in post-translational modifications play important roles in regulating the biological clock, and protein kinases may be involved in the occurrence and development of various diseases caused by biological clock disorders, including tumors. Ramanathan et al. 109 have found that inhibition of mTOR function slows circadian rhythms, thus suggesting biological regulation of mTOR in circadian rhythms. mTOR, a central regulator of multiple tumor-associated signaling pathways, plays important roles in cell proliferation, growth, differentiation, and survival. Abnormal activity of mTOR is closely associated with the occurrence, development, metastasis, and drug resistance of various malignant tumors, including lung cancer 110,111 . The PI3K/Akt/mTOR signaling pathway regulates cell growth and proliferation. Abnormal activation of this pathway is closely associated with tumor occurrence and development 112 . The AMPK/ mTOR signaling pathway is involved in tumor cell proliferation, apoptosis, invasion, metastasis, and drug resistance 110 . AMPK is an important cellular energy sensor that plays a role in metabolic control and serves as a central sensor for metabolic signals 113 . On the one hand, AMPK-mediated phosphorylation of CRYs and CK1 regulates the negative feedback control of the circadian clock through proteolytic degradation, thereby shortening the rhythm cycle 114,115 . On the other hand, most early studies have suggested that AMPK itself acts as a tumor suppressor in tumors. AMPK activation is conducive to the occurrence of various malignant tumors by inhibiting the proliferation of tumor cells, and it also promotes apoptosis by inhibiting the mTOR signaling pathway 116,117 . In summary, various protein kinases and biological clocks have close and complex relationships in tumor development.

Other possible mechanisms
In addition to the potential relationship between CLOCK and tumors, various studies have suggested potential mechanisms that may have research value. For instance, in the TME in prostate cancer, if PER expression is too low, BMAL1 expression increases, thus resulting in up-regulation of β-Catenin phosphorylation and activation of the Wnt/β-Catenin pathway. PER is a negative regulator of prostate cancer stem cells and is used as a new target for prostate cancer treatment 118,119 . In other studies, some potential targets have been proposed but have not been studied, such as iron death (colon cancer), NF-κB, REGγ, MYCN (advanced neuroblastoma), and P53 120-124 . Length constraints prevent us from stating them individually herein. These mechanisms may require additional research to guide the development and exploration of related cancer treatment issues.

Effects of CLOCK components in specific cancers
In addition to the aforementioned mechanisms, CLOCK may exhibit different biological functions and characteristics in different cancers (Figure 4). Here, we briefly discuss the roles of CLOCK/BMAL1 in in several representative cancers.

Hepatocellular carcinoma (HCC)
HCC is one of the most common malignant tumors and the fourth leading cause of cancer-associated deaths worldwide 125 . Because of its low detection, high recurrence rates, and poor prognosis in the curable stage, the survival rate of patients is unsatisfactory despite recent progress in treatment strategies 125 . In 2012, approximately 700,000 people died from liver cancer. According to the World Health Organization, more than 1 million people will die of liver cancer in the year 2030 126,127 . The main risk factors for liver cancer include hepatitis C or hepatitis B viral infection, long-term alcohol consumption, and nonalcoholic fatty liver disease (NAFLD), including the effects of smoking, obesity, diabetes, and excessive iron load 128 .
HCC, the most common type of liver tumor, is found in approximately 90% of patients with a cirrhosis history 126,129 . Previous studies have demonstrated that time-caloric restriction upregulates the expression of BMAL1, limits the transformation of liver fibrosis to cirrhosis, and further leads to HCC development, as verified in a liver cancer model induced by diethylnitrosamine 130 . These findings suggest that CLOCK may influence liver cancer occurrence by affecting liver cirrhosis progression.
Metallothionein (MT) expression controlled by CLOCK is associated with various endocrine diseases, mental diseases, and tumor diseases in humans. MT expression is relatively extensive in the liver and is regulated by metal regulatory transcription factor-1 (MTF-1) [131][132][133] . MT expression is a prognostic indicator in patients with HCC 121 . Interactive relationships among CLOCK, MT, and HCC have been identified. Li et al. 134 have confirmed that disorders in MTs and circadian clock genes play crucial roles in human HCC, possibly because of their influence on the expression of nuclear orphan receptor factor protein (Nr1d1) and D-box binding protein (DBP). Concurrently, melatonin, a drug used to treat various diseases and inhibit tumor growth [135][136][137] , prevents BMAL1 expression, an effect potentially associated with the upregulation of MT receptors and the decrease in Hif-1α 138 .
In addition, Hepatocyte nuclear factor 4α (HNF4α) is a cytokine widely present in the nuclei of hepatocytes, which is involved in the metabolic processes of various hepatocytes 139,140 . HNF4α has various physiological functions, including inhibiting expression of multiple genes (such as cyclin D1 and CCND1) in HCC development [141][142][143][144][145][146] . HNF4α expression may be negatively correlated with tumor growth, and its overexpression may inhibit liver cancer growth. Similarly, HNF4α expression is relatively lacking in human HCC 145,147 . HNF4α has 2 different promoters, P1 and P2, producing HNF4α1/2 and HNF4α7/8, respectively. Notably, this difference also exists in colon cancer 142,148,149 . According to research by Fekry and colleagues 150 , both subtypes have an apparent circadian rhythm, but P2-HNF4α directly inhibits BMAL1 expression, thereby increasing P1 isoform expression in the cytoplasm. In HNF4α-positive HCC, the increase in BMAL1 expression slows tumor growth in vivo, thus indicating that BMAL1 may be a potential target for reversing HNF4α-positive HCC.

Colorectal cancer (CRC)
CRC is a common malignant tumor. CRC-associated deaths account for approximately 10% of cancer-associated deaths worldwide and are the major cause of cancer deaths [151][152][153] . Circadian rhythm is an important factor affecting CRC occurrence and development. CLOCK inhibits early tumorigenesis in the intestines. BMAL1 affects AKT, mTOR, and P53 154 , and regulates various signal transduction pathways in intestinal stem cells (including the Hippo pathway), thus affecting CRC occurrence 155 . As described above, BMAL1 influences tumor progression by regulating the inflammatory response, in agreement with the findings of Liu et al. 156 indicating that circadian rhythm disruption leads to impaired regulatory B cell function and apoptosis of CD4 + T cells in the intestinal epithelium, and subsequently enteritis progression and CRC occurrence. Liver metastasis of CRC (CRLM) is a sign of poor prognosis in CRC, and lung and liver metastases are among the leading causes of CRC-associated deaths 157 . CLOCK expression is perturbed in CRLM model mice 158 , thus suggesting the effects of CLOCK-associated components on CRLM. BMAL1 also activates Rab27a, a key molecule associated with exosome secretion, and affects CRLM by enhancing exosome secretion in CRC cells 159 .

Breast cancer
Breast cancer is the second leading cause of cancer-associated deaths globally and the most common cancer in women 160,161 . In breast cancer cells, compared with normal breast cells, previous studies have found that the circadian rhythm and CLOCK gene expression show different degrees of damage 162,163 , and the expression of core CLOCK genes in patients with breast cancer is altered, thus confirming the role of CLOCK in breast cancer 164,165 . BMAL1 knockout promotes breast cancer cell metastasis 166 and decreases acidosis-mediated metastasis of breast cancer by decreasing hypoxia-induced acidosis in the TME 167 , thus providing a potential mechanism for preventing breast cancer metastasis.
Triple-negative breast cancer (TNBC) is a type of breast cancer with high invasiveness, poor differentiation, and ready metastasis, which accounts for about 15%-20% of all breast cancer. Owing to a lack of ER, PR, and HER-2, treatment is limited, and TNBC prognosis is poor 168 . However, studies have confirmed that acetylserotonin methyltransferase (ASMT) affects the invasiveness of TNBC cells by regulating circadian rhythms. Thus, ASMT is currently the most promising target for TNBC treatment 169 . According to recent research, BMAL1 may be directly or indirectly affected by insulin and feeding, thus resulting in changes in the characteristics of TNBC, an aspect warranting further attention 65,170 .

Glioma
Glioma is a primary tumor of the central nervous system. As described earlier, in addition to the important roles of CSCs and CD8+ T cells in glioma, CLOCK alters the content of microglia in GSC through transcriptional regulation of the chemokine olfactomedin-like 3. In the tumor microenvironment of glioma, glioma-associated microglia (GAMs) are key components regulating tumor progression 171 . GAMs are divided into 2 subtypes with opposite functions: M1 and M2, which inhibit and promote tumor progression, respectively [172][173][174] . Exosome miRNA is an important medium for communication of various biological information inside and outside cells. Li et al. 175 have used exosome-associated technologies to confirm that M2 subtype GAMs regulate the expression of tumor-associated proteins and decrease apoptosis, thereby promoting the proliferation and metastasis of glioma, which is associated with the recruitment of miR-7239-3p and a decrease in BMAL1 expression in TAMs.
The circadian clock has a wide range of effects on specific cancers. Because of space limitations, we discuss several representative cancers. Other factors such as AML 60 and melanoma 176,177 have been associated with CLOCK. However, the specific mechanisms remain unclear or controversial, and require further investigation.

Circadian clock and cancer treatment
We have described the complicated mechanisms underlying the relationship between circadian rhythm and cancer. To some extent, these mechanisms are believed to be valuable in guiding cancer treatment. Both in vivo and in vitro experiments and statistical studies on clinical cases have shown that biological CLOCK has important research value in treating tumors or regulating the anti-tumor efficiency of radiotherapy and chemotherapy [178][179][180][181] . Several aspects are summarized below.

CLOCK affects the sensitivity of tumor cells to treatment and the efficacy of drugs
As described previously, CLOCK affects the occurrence, development, and metastasis of CRC. The standard chemotherapy agents 5-fluorouracil and bevacizumab (Beva) are often used for metastatic CRC. Bevacizumab inhibits angiogenesis by preventing the binding of vascular endothelial growth factor A (VEGFA) to its receptor, thereby achieving antitumor therapeutic effects. However, it is effective for only some clinical patients, and its anti-angiogenic effect is adaptive 182,183 , thus suggesting the possibility of drug resistance. BMAL1 upregulates the promoter activity of VEGFA and enhances its expression, as confirmed by BMAL1 inhibition [184][185][186] . In addition, VEGF expression in xenografts (sarcomas, melanomas, etc.) is induced by hypoxia. Circadian rhythms are inhibited by transcription of PER2 and CRY1. Therefore, ZT2 is more effective in anti-angiogenesis therapy than ZT14 (where ZT0 is bright, and ZT12 is dark) 184 . After administration of Beva and other anti-angiogenic drugs, BMAL1 expression in cancer cells increases, thus leading to the activation of VEGF expression and inducing drug resistance 187 . These results emphasize the roles of CLOCK in influencing tumor angiogenesis and anti-angiogenic drug resistance.
Epithelial-mesenchymal transition (EMT) refers to the polarization of epithelial cells to obtain characteristics of mesenchymal cells. This process enhances the migration and invasion of cells, and mediates drug resistance. EMT is the first key step in metastatic CRC development 188 . In the formation and maintenance of EMT, the downregulation of E-cadherin expression causes functional disorders of the E-cadherin/βcatenin complex, and various signaling pathways (such as TGFβ and Wnt) are involved 189,190 . A recent report 191 has indicated that BMAL1 is critical in EMT-induced CRC metastasis and drug resistance to chemotherapeutic drugs. Understanding the importance of BMAL1 in the epithelial-mesenchymal equilibrium of CRC cells will aid in studying how to reverse drug resistance to existing treatment methods. Similar findings have been reported for breast cancer and glioma.
A study on MCF10A (non-tumorigenic) and MDA-MB-231 (invasive-tumorigenic) cells with 2 different tumorigenic characteristics of human breast epithelial cells has found that knockout of BMAL1 increases sensitivity to cisplatin and doxorubicin 23 . Temozolomide (TMZ) is a DNA alkylator commonly used to treat GBM. When the effect of TMZ on GBM (including DNA damage, cell death and tumor growth inhibition) reaches the maximum daily peak of BMAL1 expression, the anti-tumor effect of TMZ has circadian regularity, which may be associated with differing sensitivity of GBM cells to TMZ, owing to their circadian regularity 192 . Similarly, in a study of GBM cells, after bortezomib treatment, cell viability has been found to change with time, and this rhythm is altered by BMAL1 knockdown 193 .
In summary, the destruction of BMAL1 usually increases the sensitivity of tumor cells to chemotherapeutic drugs; however, some studies have indicated opposite results. For example, in pancreatic cancer 194 , BMAL1 is targeted and inhibited by miR-135b, thus resulting in local circadian rhythm disorders. However, this destruction has been found to cause tumor resistance to chemotherapeutic drugs, in contrast to previous conclusions. Similarly, in tongue squamous cell carcinoma, BMAL1 overexpression increases sensitivity to paclitaxel, whereas decreasing the expression of BMAL1 induces drug resistance in tongue squamous cell carcinoma cells 195 .
Some researchers have proposed that 1A-116 in GBM treatment, at the end of light (ZT12) administration rather than the beginning of light (ZT3) administration, prolongs survival time in mice 196 . This finding is contrary to the conclusion that at ZT2, the time of administration in CRC, anti-angiogenesis drugs have a more potent anti-tumor effect. Treatment with 1A-116 specifically inhibits the activation of Ras-related C3 botulinum toxin substrate 1 (RAC1) in GBM cells, thereby affecting the role of RAC1 in tumor progression. The mechanism of action of 1A-116 may block the binding of RAC1 to guanine exchange factor by interacting with Trp56 residues and interfering with the normal biological function of  . This contradictory phenomenon requires further research.
Beyond increasing the sensitivity of tumor cells to anti-tumor drugs, some drugs, such as doxorubicin, destroy circadian cell rhythms by inhibiting BMAL1 expression 201 . Thua, targeting BMAL1 may be a feasible strategy for improving the therapeutic effects of anti-tumor drugs. These CLOCK-mediated differences in the sensitivity of anti-cancer drugs may be used to optimize and adjust treatment regimens and medication protocols to achieve the best anti-tumor effect. However, BMAL1 has a bidirectional role: decreasing BMAL1 expression increases the sensitivity of cells to anticancer drugs, but also enhances the invasiveness of tumors and causes distant metastasis. Therefore, this conclusion should be comprehensively considered.

Potential therapeutic targets or drugs
We described some potential mechanisms through which CLOCK affects cancer progression. The components associated with these mechanisms have the potential to be used as targets for cancer treatment. Some drugs may have anti-tumor effects by regulating circadian rhythms, as summarized below.

Ghrelin
Cachexia occurs in most cancer patients, particularly in the terminal stage of cancer and in patients with metastasis. It is a severe cancer complication that directly affects patients' tolerance to treatment, and can even cause disability or death [202][203][204] . More than half of all patients with cancer are affected by cachexia, and at least 20% of patients with advanced cancer die 205 . As described above, Ghrelin, an intestinal hormone, activates and promotes appetite and affects glucose metabolism, gastric acid secretion, and other physiological activities. Consequently, it is often considered to reverse cancer-associated cachexia and to be a promising treatment [206][207][208][209] . The rhythmic characteristics of Ghrelin secretion may provide a more optimized solution for future clinical applications.

CLK8
According to Doruk et al. 210 CLOCK's interaction with small molecules (CLK8) decreases the interaction between CLOCK and BMAL1 and interferes with the translocation of CLOCK into the nucleus. This decrease in CLOCK in the nucleus leads to a more stable negative arm of TTFL and enhances the role of the circadian clock. Therefore, by influencing the functions of CLOCK and BMAL1, CLK8 elicits improvements in aging, emotional disorders, and the effectiveness of cancer treatment. Another small molecule, Nobiletin, a natural polymethoxy flavonoid compound, may also enhance the CLOCK effect by increasing the PER2 level and consequently stabilizing the negative arm of TTFL.
The above small molecule substances may enhance the functions of CLOCK and BMAL1, and are the leading compounds and potential targets for preventing and treating new therapies for diseases associated with circadian rhythm changes, such as cancer. mTOR mTOR is a highly conserved serine/threonine-protein kinase associated with cell proliferation, protein metabolism, and autophagy [211][212][213][214] . The mTOR signaling pathway is abnormally expressed in various tumors. Although mTOR inhibitors have broad application prospects in antitumor therapy, problems remain, such as low treatment efficiency and easy recurrence.
Ramanathan et al. 109 have found that inhibition of mTOR function slows circadian rhythms, thus suggesting biological regulation of mTOR in circadian rhythms. The PTEN gene inhibits tumor growth by negatively regulating PI3K and its downstream targets (mTOR/AKT, etc.). PTEN dysfunction is observed in melanoma, breast cancer, liver cancer, and other cancers. According to previous research 215 , targeting PTEN upregulates BMAL1 expression, whereas this effect is reversed by rapamycin (mTOR inhibitor). The accumulation of BMAL1 caused by PER deficiency is also eliminated by rapamycin. Thus, mTOR-Bmal may be a potential clinical target. Combined biological rhythm and anti-mTOR drugs may further improve the anti-tumor effects of traditional anti-mTOR drugs.

UPR
Metabolic disorders, viral infection, hypoxia, and tumors in the body usually cause UPR, and irreparably damaged cells may undergo apoptosis, on the basis of decreased protein synthesis. The UPR is often involved in tumor occurrence and development. As described above, it promotes the survival of tumor cells, which may be associated with the inhibition of BMAL1 expression by miR-211 induced by PERK 87,216-218 . Therefore, PERK and Ire1, as UPR transducers, are potential targets for treating tumor progression.

HSP90
The expression of some HSP90 isomers may have a circadian rhythm. Although the amplitude of this rhythm is low, it nonetheless affects the cell cycle process and may lead to time-dependent anti-tumor efficacy of some drugs. Therefore, targeting HSP90 also improves therapeutic effects 219 .

MLN4924
Osteosarcoma is a malignant bone tumor that occurs primarily in adolescents. Surgery after neoadjuvant chemotherapy is a conventional treatment, and adjuvant chemotherapy (cisplatin, doxorubicin, etc.) is performed after surgery. The 5-year survival rate remains low 220,221 . Nedd8 is a ubiquitin-like molecule with a molecular weight of approximately 8 kDa whose substrate is cullin protein. Cullin protein, as the scaffold of Cullin-Ring E3-ubiquitin ligases, participates in the degradation of cellular proteins in the ubiquitin-proteasome system. MLN4924 (Pevonedistat) is an inhibitor of Nedd8 activation enzyme, which has anti-cancer effects and inhibits the occurrence and development of tumors [222][223][224][225][226][227] . According to Zhang et al. 228 , MLN4924 is an effective drug for treating osteosarcoma and has vast application prospects. RORα and BMAL1 may mediate its inhibitory effect on tumor growth.

Focus on therapeutic approaches associated with circadian-mediated immune responses
Lymphocytes often play important roles in antitumor immunity, and their biological activity is often regulated by CLOCK. Many cytokines with immune activity have a clear circadian rhythm during their production or secretion.

Figure 5 Continued
For example, CD4 + 17 helper T cells (Th17) produce interleukin-17 (IL-17), and Th17 differentiation is regulated by the transcription factor RORγT, which has a circadian rhythm 229 . RORγ activation enhances the cell differentiation and biological function of Th17 and decreases the Treg level (But Treg itself has no circadian rhythm 230 ), thus inhibiting tumor growth. This antitumor effect has also been verified in animal models 231 . We also suggest that the circadian clock affects antitumor immunity by regulating immune-associated functions, such as depletion of T cells, enhancement of immune escape, and controlling the metabolic process of TAMs by affecting the function of Hif-1α. Therefore, focusing on these immune responses associated with CLOCK and BMAL1 should provide new insights into cancer treatment.
To better understand the role of circadian-mediated immunity in cancer progression, we performed a correlation analysis, and the results are depicted in Figure 5A. TMB reflects the density of the non-synonymous mutation distribution in protein-coding regions, usually represented by the total number of somatic tumor mutations in each MB tumor genome region. Tumors with high TMB may have more neoantigens that the immune system recognizes. As displayed in Figure 5B, in different cancers, BMAL1 has different or even opposite effects on the function of the immune system, in agreement with the contradictory points proposed above. Therefore, specific analyses should be performed for specific types of cancer.    Contradictions remain in the current research on the underlying mechanisms. However, the circadian clock as a target is not affected by the lack of mechanistic uncertainty, and this field has great research value in oncology, for example to provide prognostic indicators for cancer progression (Figure 6). This article summarized many possible effective targets for tumor therapy and provides a reference for related research. Further modification of small-molecule compounds known to regulate the biological clock, optimization of their pharmacokinetic properties, and minimization of adverse reactions are important research directions in this field. At present, research on most small molecule regulators remains in basic experimental stages, and biological clock regulating compounds with good therapeutic effects and pharmaceutical characteristics in animal models must enter the next stage of clinical trials.
In addition, owing to the rhythmic expression of circadian clock genes, the optimal administration times for clock-targeted drugs and chemoradiotherapy are considered to maximize anti-tumor effects and minimize toxic and adverse effects. For example, treating cancer by administering mTOR inhibitors as at the peak of mTOR expression improves the survival rate in mice 232,233 . These unclear mechanisms are likely to gradually be elucidated in the future, thus deepening understanding of the role of CLOCK and BMAL1, and providing new ways to treat cancer.

Conflict of interest statement
No potential conflicts of interest are disclosed.