Insights about circadian clock in glioma: From molecular pathways to therapeutic drugs

Abstract Glioma is characterized as the most aggressive brain tumor that occurred in the central nervous system. The circadian rhythm is an essential cyclic change system generated by the endogenous circadian clock. Current studies found that the circadian clock affects glioma pathophysiology. It is still controversial whether the circadian rhythm disruption is a cause or an effect of tumorigenesis. This review discussed the association between cell cycle and circadian clock and provided a prominent molecular theoretical basis for tumor therapy. We illustrated the external factors affecting the circadian clock including thermodynamics, hypoxia, post‐translation, and microRNA, while the internal characteristics concerning the circadian clock in glioma involve stemness, metabolism, radiotherapy sensitivity, and chemotherapy sensitivity. We also summarized the molecular pathways and the therapeutic drugs involved in the glioma circadian rhythm. There are still many questions in this field waiting for further investigation. The results of glioma chronotherapy in sensitizing radiation therapy and chemotherapy have shown great therapeutic potential in improving clinical outcomes. These findings will help us further understand the characteristics of glioma pathophysiology.


| INTRODUC TI ON
Glioma is characterized as the most aggressive brain tumor type with a median survival period of only 14 months and a 5 years survival rate of less than 10% after diagnosis. 1-4 Current standard treatment strategies include complete or sub-complete surgical resection, alkylating agent administration chemotherapy, and radiation therapy. 5,6 Alternative treatment can also be applied, including immunotherapy, targeted therapy, and tumor treatment fields (TT fields). [7][8][9] Since glioma is characterized by rapid proliferation and aggressive infiltration, a considerable proportion of patients develop local recurrence and central nervous system metastasis. 10 The circadian rhythm is an essential cyclic oscillation system generated by the endogenous circadian clock. 11,12 Essentially all mammalian organisms have developed circadian rhythms in order to synchronize social and physiological capabilities with explicit periodicity. 13,14 The control center of this circadian rhythm is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which regulates the rhythm production of various physiological functions. 15,16 Accumulating evidence reveals that the circadian rhythm has an impact on glioma pathophysiology. Temozolomide (TMZ) administration in the morning rather than evening exhibited longer overall survival (OS) with a significant first-year restricted mean survival time difference. 17 Lowdose Bortezomib, the proteasome inhibitor in anticancer therapy, displayed higher efficacy when administered in tumor-bearing animals at night compared to day/night administration. 18 In mice injected with circadian clock gene Bmal1 knocked-down cells, a higher tumor growth rate was observed. 18 Khan et al. revealed glioma-genesis gene expression changes in the mouse brain after chronic alternating lightdark cycle exposure and suggested a potential connection between circadian clock disruption and glioma genesis risk. 19 Wang F et al. revealed circadian clock disturbance promotes carcinogenesis through circadian clock gene Period 2 (Per2) deregulation. 20 Glioma also participates in circadian alteration and disruption. Athymic nude mice implanted with LN229 human glioma cells showed an increase in the endogenous period of the circadian clock and a slower resynchronization rate. 21 SCN astrocytes modulate the circadian pacemaker via the regulation of glutamate levels. 22,23 Meanwhile, increased glutamate levels are characteristic of glioma, suggesting that dysregulation of proper glial function occurring in malignant tissue may impact timekeeping and clock synchronization. 24,25 In addition, molecules including TNFα and CCL2 involved in immune response affect the core circadian oscillator. [26][27][28] Glioma alters the microenvironment to resist immune attack and cytokines and chemokines can be hijacked and then lead to circadian disruption. 26,29 Circadian activities associated with cerebrovascular reactivity and brain energy metabolism help to maintain central nerve system homeostasis. 30,31 Cerebral arteries possess a functional circadian clock and exhibit a diurnal rhythm in vasoreactivity to ATP. 30 Brain metabolites are altered during sleep including acylcarnitines, hydroxylated fatty acids, phenolic compounds, and thiol-containing metabolites. 31 Disruption of these oscillations has been observed in cerebrovascular diseases. [32][33][34] However, the circadian activities of glioma are still poorly understood, and circadian rhythm disruption is still controversial whether it is a cause or an effect of glioma genesis.
Herein, we discussed the molecular connection between cell cycle and circadian rhythm clock, which are the major cyclic system, and focused on the external factors and internal characteristics associated with the glioma circadian clock. This review also summarized the molecular pathways and therapeutic drugs in glioma treatment by reviewing the circadian clock concerning differential molecular changes to explore the potential value in translational research.

| CELL C YCLE AND CIRC AD IAN CLO CK
Currently, the chemotherapeutic alkylating agent TMZ and radiation therapy regulate and inhibit cellular proliferation by destructing DNA replication. 35,36 DNA damage triggers the activation of cell cycle checkpoint pathways, which cause the cell cycle to be suspended, so the DNA repair machinery can detect and repair the damage. 37 The DNA damage response (DDR) maintains genomic integrity while contributing to the resistance to chemotherapy and chemotherapy. [36][37][38] The cell cycle checkpoint is an important cellular mechanism that prevents uncontrolled proliferation. 39 The glioma cell cycle circulation has been well characterized, which consists of the rest/growth phase (G0/G1 phase), DNA synthesis phase (S phase), G2 phase, and mitosis phase (M phase). 40 Arresting at G1/S or G2/M phase can effectively inhibit the cell proliferation process. 41 The core cell-cycle regulation mechanism depends on the cyclin-dependent kinase (CDK) activation. 42 The circadian clock oscillations rely on transcriptionaltranslational auto-regulatory feedback loops (TTFL), which are regulated by the activity of core molecular components. 43 In mammals, the core clock genes govern circadian rhythm and consist of circadian locomotor output cycles kaput (Clock), brain and muscle ARN-t like protein 1 (Bmal1), neuronal PAS domain protein 2 (Npas2), period protein family (Per1, Per2, and Per3), and cryptochrome family (Cry 1 and Cry 2). 44 The clock control genes include differentially expressed in chondrocyte family (Dec1 and Dec2), nuclear receptor subfamily 1, group D member 1(REV-ERBα), retinoic acid receptor-related orphan receptor α (RORα), casein kinase 1 family (CKIε and CKIδ), and timeless (Tim). 44 These circadian clock genes encode a highly conserved alkaline helix-loop-helix (bHLH) region that binds to DNA sequence "CANNTG" (E-Box), which can be used as a core transcription enhancer. 45 Conserved core components include the bHLH-PAS transactivators, CLOCK/NPAS2, and BMAL1. 46 PERs and CRYs inhibit CLOCK: BMAL1 heterodimer transcriptional activity and in turn their own expression, thus forming the negative feedback loop. 47 An additional loop that is also induced by CLOCK: BMAL1 heterodimerization activates the rhythmic transcription of Rev-erbα and Rorα. 48 In addition, the CLOCK: BMAL1 heterodimer complex may be involved in activating a second alternative cycle consisting of REV-ERBα and REV-ERBβ which compete at the ROR-binding elements. 49 CKIε and CKIδ determine the circadian period length, through speed and rhythmicity regulation of PER1 and PER2 phosphorylation. 50 DECs provide feedback and modulate CLOCK activity. 51 Circadian rhythms persist in the presence or absence of environmental cycles because they are generated by endogenous mechanisms. Their periodic activity is composed of a periodically oscillating network formed by a series of rhythmically expressed proteins, and a variety of circadian clock factors affect the cell cycle by regulating the expression of the cyclin. In the G/S phase, REV-ERBα inhibits p21 to promote cell progression, 52 RORα activates p21 to inhibit cell progression, 53 DEC1 inhibits cyclin D1, 54 and CLOCK/BMAL1 negatively regulates c-Myc 55 ; in the G/M phase, BMAL1/CLOCK, BMAL1/NPAS2 or CRY1 acts on Wee1 to inhibit or activate glioma process 56 (Figure 1). Therefore, elucidating the circadian rhythm effect on the cell cycle will provide a prominent molecular theoretical basis for tumor therapy.

| Thermodynamics
Temperature compensation is characterized as the robust output of the circadian rhythm to the temperature fluctuation. 57 Since the system level is temperature-stable while individual components in the system are usually temperature-sensitive under varying temperatures, the temperature compensation mechanism is peculiar. 58 The Cry1 gene expression fluctuated with the temperature while the other circadian genes showed no significant change. 58 The temperature amplitude showed a tight connection between circadian and metabolic rhythms.

| Hypoxia
Circadian dysregulation is exacerbated within the hypoxic tumor microenvironment (TME) in glioma. 59,60 The hypoxia-inducible factor-1α targets were negatively correlated with tumor suppressor Clock gene. 61,62 Loss of fidelity in timekeeping accelerates tumor development. 63 Timekeeping fidelity loss or gain mechanism may offer a controllable switch for pharmaceutical research. [61][62][63] Exploring interactions between the hypoxia and the circadian clock in TME may achieve a therapeutic advantage in hypoxia-modifying compounds combined with first-line treatments.

| Post-modification
Chromatin modification and post-translational modification (PTM) of protein have a great impact on maintaining the periodic oscillation of the 24 h rhythm. 63 Chromatin modification mainly regulates the transcriptional oscillation of rhythmic genes through histone acetylation, deacetylation, and methylation on the promoters of core clock genes. 64 The Clock gene itself has acetyltransferase activity and can activate the transcription of the core clock gene through the CLOCK: BMAL1 heterodimer 65 ; on the contrary, the inhibition of the core clock gene through the PERs/ CRYs heterodimer is through regulating histone deacetylation and methylation. 66 Phosphorylation, acetylation, and ubiquitination of protein PTM regulate activation of circadian oscillation. [65][66][67] Tyrosine kinases CKIε and CKIδ regulate nucleus/cytoplasm transfer through phosphorylation of the core clock elements. 68 CLOCK acetyltransferase activity can act on the lysine residues of BMAL1 to regulate the BMAL1 acetylation, which in turn influences the CLOCK: BMAL1 heterodimer combination. 69 Protein ubiquitination regulates the core clock proteins' stability and thus affects circadian rhythms. 70 IRE1a endoribonuclease could lead to PER1 degradation through cleaving the Per1 mRNA. 71 Differential expression of circadian genes in cancer or normal cells may thus provide a molecular theoretical basis for glioma chronotherapy.

| MicroRNA
Small non-coding RNAs, including MicroRNAs, participate in glioma pathophysiology processes including proliferation, invasion, survival, angiogenesis, and cancer metastasis. 72 There have been reports that miR-124 was downregulated in glioma. 73 MiR-124 decreasing may be responsible for Clock gene expression increasing, indicating its potential therapeutic values in glioma chronotherapy. 73 miR-7239-3p secreted by M2 microglial exosomes is recruited in the TME. 74 MiR-7239-3p in M2 microglial exosomes, not M1 type, inhibits Bmal1 expression, promotes proliferation, and reduces apoptosis of glioma cells. 74  phase activation in the Per2 gene is a potential target for treatments that may suppress EMT, minimize GSCs, and limit tumor metastasis. 77 Per2 gene expression was enriched within C6 glioma tumor spheres but not in monolayer cell culture, suggesting that cell interactions or TME enable circadian timing. 51

| Metabolism
The molecular circadian clock circadian showed a tight connection with the metabolic/redox oscillator, which presents in organs, tissues, and individual cells. 46 The molecular rhythm disruption may cause metabolic disorders. 46 The synthesis and degradation of glycerophospholipids (GPLs) exhibit an oscillation in metabolisms with a 24 h periodicity. 78 Moreover in quiescent cells, the Per1 gene is tightly involved in GPL synthesis regulation. 79 While in proliferating cells, targeting time-dependent high-level redox and low-level GPL state revealed a potential efficient therapeutic pathway in chemotherapy. 80 Constant light, which leads to circadian disruption, promotes anabolic metabolism. 81 Under circadian disruption, more macrophages were recruited in the TME, genes involved in lipogenesis, and glucose uptake was upregulated. 79,81

| Chemotherapy sensitivity
The chemotherapeutic alkylating agent TMZ and radiation therapy regulate and inhibit cellular proliferation by destructing DNA replication. 35

| Radiation therapy sensitivity
PERs participate in regulating the circadian rhythm in mammalian organisms. High Per1 and Per2 expression were associated with increased sensitivity to irradiation in glioma tissue. 83 Following exposure to irradiation, higher Per1 expression levels lead to serious DNA damage while the expression of important checkpoints in DNA damage, such as CHK2 and P53, increased. 84 Cry2 mRNA and protein levels exhibit 8 h periodicity in glioma tissue compared to 24 h in normal brain tissue. Higher Cry2 expression in glioma tissues was in association with increased cell proliferation and irradiation resistance. 85 Silencing Clock downregulated c-Myc and Cyclin B1 and led to apoptosis and cell cycle arrest after irradiation. 86

| THE MOLECUL AR PATHWAYS INVOLVED IN G LI OMA CIRC AD IAN CLO CK
The key molecules of the circadian clock are involved in proliferation, invasion, migration, and tumorigenesis. The pathways involving circadian clock-related molecules are summarized in Table 1.

| PI3K/AKT pathway
Protein kinase B (PKB/Akt) activation via phosphatidylinositol 3-kinase (PI3K) is highly related to tumorigenesis. 87 In glioblastoma cells, the PI3K pathway regulates Cry expression. CRY is necessary and sufficient to promote the accumulation of oncogene Myc. 88 PI3K/AKT pathway could also be regulated via tyrosine kinase AXL, which is the transcriptional target of REV-ERBβ in glioblastoma cells. 89 The core circadian clock gene Bmal1 knockdown elevated cell invasion and migration through phosphorylated-AKT and matrix metalloproteinase-2 (MMP-2) accumulation. 90 BMAL1 is characterized as a tumor suppressor, which is capable of suppressing cancer cell growth and invasiveness. 91 Recent studies demonstrated that there is a tight molecular connection between circadian rhythms and glioma genesis via PI3K/AKT pathway.

| TGFβ/Smad
The TGFβ pathway participates in many cellular processes, including cell proliferation, invasion, migration, and extracellular matrix remodeling. 92

| Wnt/β -catenin
The WNT/beta-catenin pathway induces the genes transcription involved in cell proliferation, cell invasiveness, nucleotide synthesis, tumor growth, and angiogenesis. 95 WNT/β-catenin signaling upregulation also induces molecular differential changes in core metabolic enzymes that modify their thermodynamics behavior. 96 This leads to pyruvate dehydrogenase kinase 1 (PDK1) and monocarboxylate lactate transporter 1 (MCT1) activation. 96 Consequently, phosphorylation of PDK1 inhibits the pyruvate dehydrogenase complex, which leads to aerobic glycolysis despite the oxygen availability, named the Warburg effect. 96 In glioma cells, the Wnt/β-catenin signaling pathway was identified as the target of PER2 in GSCs. 97 Subsequently, downstream molecular PPARγ is downregulated, resulting in abnormalities in the regulation of circadian rhythms and destruction of circadian clock genes. 98 These results indicated that PER2 plays a critical role in regulating the stemness of GSCs via the WNT/β-catenin signaling pathway.

| TP53
The MDM2/TP53 pathway is an important pathway for the occurrence and development of tumors. It is well characterized that TP53 is an important tumor suppressor gene. 99

| P38 MAPK
The p38 MAPK expression and activity increase are correlated with poor clinical prognosis, including GBM multiforme; however, the lethal toxicity of p38 MAPK inhibitors limits their therapeutic use. 104 The phosphorylated p38 MAPK protein level was reduced in Clockdeficient cells. 104 The p38 MAPK activity inhibition with specific inhibitor VX-745 led to cell-type-specific periodical changes in the molecular clock, indicating potential therapeutic use of VX-745 in glioma chronotherapy. 104 Under the control of the circadian clock, the p38 MAPK activity inhibition in invasive IM3 cells at the time point when the levels are normally low in human astrocytes significantly reduced IM3 invasiveness. 104

| Lactate-IL-1β -Clock Loop
A desynchronized circadian rhythm in tumors is coincident with aberrant inflammation and dysregulated metabolism. 105 The increase in tumor metabolite lactate results in the cytokine interleukin-1β (IL-1β) upregulation leading to inflammatory. 106 IL-1β was correlated with elevated levels of the core circadian regulators Clock and

| Curcumin
Curcumin (diferuloylmethane, CAS#:458-37-7) is a promising phytochemical that can be administered in glioma therapy. 121 Administration of curcumin can alter molecular circadian timing within cells. The prominent target, BMAL1, is the core gene in molecular oscillators that generates circadian rhythms. 122 Studies reported that curcumin can affect STAT, PPARγ, and NF-kB expression within two interacted molecular timing loops. 121,122 BMAL1 is activated by curcumin via PPARγ stimulation. 108 Research has also shown that curcumin activates sirtuin 1, and binds to CLOCK: BMAL1 heterodimer to promote the deacetylation and degradation of PER2. 69 Although no effect on the circadian mechanism has been reported, 10 μM curcumin treatment did disrupt a single circadian oscillator within the clock unit or coupling between circadian clocks in apoptosis. 123 During anticancer treatment, curcumin or its analogs should be administered to tumor cells at the optimal stage in maximizing efficacy after determining the circadian phase.

| Norepinephrine
Norepinephrine (NA, CAS#: 51-41-2) is primarily located in the brain stem and is involved in behaviors including sleep and awakening. 124 NA can act on the biological pineal region cells pinealocytes. to the environmental cycle of day and night. 21,112 Besides, the different TME between in vivo and in vitro are worthy of attention.
The circadian clock of laboratory animals may differ significantly from those of humans. Since mice are nocturnal animals, and their active and rest phases are out of phase with humans, there are obvious differences in circadian rhythms in mice and humans. 130 In addition, tumor cell-intrinsic circadian rhythms can regulate TMZ cytotoxicity in mice, which showed a strong correlation between drug sensitivity and circadian rhythm, 82 while the effect of TMZ in glioma chronotherapy treatment is still controversial and needs further large-population-based trials for validation. 17,123 Despite being more complex and expensive than murine models, nonhuman primates share more closely activity patterns with humans, which may provide stronger evidence support for chronotherapy research. 131 The generalization of these conclusions to other species will require additional systematic study. Besides, several non-invasive imaging modalities, including quantitative parametric images of O-

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.