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Circadian-driven transcriptional programs govern metastatic progression

Jie Wang, Hao Pan, Xuan Wang, Yuru Ren, Yiwen Huang, Yizhou Liu, Hejin Lai, Zhimin Fei, Ning Pu and Yu Wang
Cancer Biology & Medicine September 2026, 23 (9) 1213-1229; DOI: https://doi.org/10.20892/j.issn.2095-3941.2025.0491

Article Figures & Data

Figures

  • The TTFL of circadian rhythm. The circadian clock is organized around the TTFL composed of three interlocked regulatory loops that coordinate rhythmic gene expression across the day–night cycle. 1. Core feedback loop: During the daytime, the heterodimeric transcription factors, CLOCK and BMAL1, bind to E-box elements in the nucleus, activating the transcription of core CLOCK genes, including PERs and CRYs, as well as CLOCK-controlled genes, such as DBP, RORs, and REV-ERBs. Newly synthesized PER and CRY proteins accumulate in the cytoplasm during the night, where CK1 phosphorylates PER proteins, regulating the stability and timing of nuclear entry. Phosphorylated PER/CRY complexes translocate into the nucleus and inhibit CLOCK/BMAL1-mediated transcription. Subsequently, PER and CRY proteins are ubiquitinated and degraded by the proteasome, thereby relieving negative feedback and allowing a new circadian cycle to begin. 2. Secondary feedback loop: In the secondary feedback loop, CLOCK/BMAL1-driven transcription induces the nuclear receptors, REV-ERBs and RORs, which competitively bind to same ROREs in the promoters of circadian genes. Through RORE-dependent mechanisms, REV-ERBs repress whereas RORs activate BMAL1 transcription, thereby stabilizing oscillatory amplitude and phase. Beyond regulating core clock components, RORE-mediated signaling contributes to the phase-aligned expression of downstream clock-associated regulators, including NFIL3, providing a temporal connection between the secondary feedback loop and D-box-dependent circadian output. 3. Third feedback loop: The loop involves DBP and NFIL3, which competitively bind to D-box elements to regulate rhythmic transcription of downstream clock-controlled genes, thereby contributing to circadian output that is temporally coordinated with ROR-mediated regulation. BMAL1, brain and muscle ARNT-like protein1; CK1, casein kinase 1; CLOCK, circadian locomotor output cycles kaput; CRYs, cryptochromes, CRY1, CRY2; DBP, d-element-binding protein; NFIL3, nuclear factor interleukin 3; PERs, period circadian regulators, PER1, PER2, PER3; RORs, retinoic acid receptor-related orphan receptors, RORα, RORβ, RORγ; RORE, ROR response element; REV-ERBs, nuclear receptor subfamily 1 group d members, REV-ERBα, REV-ERBβ; TTFL, transcription–translation feedback loop.
    Figure 1

    The TTFL of circadian rhythm. The circadian clock is organized around the TTFL composed of three interlocked regulatory loops that coordinate rhythmic gene expression across the day–night cycle. 1. Core feedback loop: During the daytime, the heterodimeric transcription factors, CLOCK and BMAL1, bind to E-box elements in the nucleus, activating the transcription of core CLOCK genes, including PERs and CRYs, as well as CLOCK-controlled genes, such as DBP, RORs, and REV-ERBs. Newly synthesized PER and CRY proteins accumulate in the cytoplasm during the night, where CK1 phosphorylates PER proteins, regulating the stability and timing of nuclear entry. Phosphorylated PER/CRY complexes translocate into the nucleus and inhibit CLOCK/BMAL1-mediated transcription. Subsequently, PER and CRY proteins are ubiquitinated and degraded by the proteasome, thereby relieving negative feedback and allowing a new circadian cycle to begin. 2. Secondary feedback loop: In the secondary feedback loop, CLOCK/BMAL1-driven transcription induces the nuclear receptors, REV-ERBs and RORs, which competitively bind to same ROREs in the promoters of circadian genes. Through RORE-dependent mechanisms, REV-ERBs repress whereas RORs activate BMAL1 transcription, thereby stabilizing oscillatory amplitude and phase. Beyond regulating core clock components, RORE-mediated signaling contributes to the phase-aligned expression of downstream clock-associated regulators, including NFIL3, providing a temporal connection between the secondary feedback loop and D-box-dependent circadian output. 3. Third feedback loop: The loop involves DBP and NFIL3, which competitively bind to D-box elements to regulate rhythmic transcription of downstream clock-controlled genes, thereby contributing to circadian output that is temporally coordinated with ROR-mediated regulation. BMAL1, brain and muscle ARNT-like protein1; CK1, casein kinase 1; CLOCK, circadian locomotor output cycles kaput; CRYs, cryptochromes, CRY1, CRY2; DBP, d-element-binding protein; NFIL3, nuclear factor interleukin 3; PERs, period circadian regulators, PER1, PER2, PER3; RORs, retinoic acid receptor-related orphan receptors, RORα, RORβ, RORγ; RORE, ROR response element; REV-ERBs, nuclear receptor subfamily 1 group d members, REV-ERBα, REV-ERBβ; TTFL, transcription–translation feedback loop.

  • Circadian clock-specific TME that affects immune suppression and angiogenesis. Schematic illustration of how circadian clock components modulate immune suppression and angiogenesis in the TME. (A) Circadian regulation of immune suppression. Core clock genes, including BMAL1, BMAL2, CLOCK, PER2, RORα, and RORγ, influence the differentiation, function, and metabolic state of multiple immune cell populations within the TME. Circadian regulation affects CAFs, CD8+ T cells, CD4+ T cells, Tregs, DCs, and neutrophils, thereby shaping immunosuppressive signaling pathways, such as NF-κB and metabolic processes, including cholesterol metabolism. In parallel, circadian disruption, exemplified by CJL, promotes the accumulation and suppressive activity of MDSCs through the CXCL5–CXCR2 axis and impairs NK cell function via altered CD122 signaling, collectively enhancing immune suppression. (B) Circadian regulation of angiogenesis. Circadian clock components, including CLOCK, BMAL1, PER2, and RORα, regulate angiogenic processes by modulating VEGF signaling in ECs. Disruption of circadian rhythms, such as exposure to LL, enhances VEGF-driven angiogenesis, thereby facilitating tumor vascularization. BMAL1, brain and muscle ARNT-like protein1; BMAL2, brain and muscle ARNT-like protein2; CAFs, cancer-associated fibroblasts; CJL, chronic jet lag; CXCL5, C-X-C motif chemokine ligand 5; CXCR2, C-X-C motif chemokine receptor 2; DCs, dendritic cells; ECs, endothelial cells; LL, constant light; MDSCs, myeloid-derived suppressor cells; NK cells, natural killer cells; PER2, period circadian regulator 2; RORα/γ, retinoic acid receptor-related orphan receptor alpha/gamma; TME, tumor microenvironment; Tregs, regulatory T cells; VEGF, vascular endothelial growth factor.
    Figure 2

    Circadian clock-specific TME that affects immune suppression and angiogenesis. Schematic illustration of how circadian clock components modulate immune suppression and angiogenesis in the TME. (A) Circadian regulation of immune suppression. Core clock genes, including BMAL1, BMAL2, CLOCK, PER2, RORα, and RORγ, influence the differentiation, function, and metabolic state of multiple immune cell populations within the TME. Circadian regulation affects CAFs, CD8+ T cells, CD4+ T cells, Tregs, DCs, and neutrophils, thereby shaping immunosuppressive signaling pathways, such as NF-κB and metabolic processes, including cholesterol metabolism. In parallel, circadian disruption, exemplified by CJL, promotes the accumulation and suppressive activity of MDSCs through the CXCL5–CXCR2 axis and impairs NK cell function via altered CD122 signaling, collectively enhancing immune suppression. (B) Circadian regulation of angiogenesis. Circadian clock components, including CLOCK, BMAL1, PER2, and RORα, regulate angiogenic processes by modulating VEGF signaling in ECs. Disruption of circadian rhythms, such as exposure to LL, enhances VEGF-driven angiogenesis, thereby facilitating tumor vascularization. BMAL1, brain and muscle ARNT-like protein1; BMAL2, brain and muscle ARNT-like protein2; CAFs, cancer-associated fibroblasts; CJL, chronic jet lag; CXCL5, C-X-C motif chemokine ligand 5; CXCR2, C-X-C motif chemokine receptor 2; DCs, dendritic cells; ECs, endothelial cells; LL, constant light; MDSCs, myeloid-derived suppressor cells; NK cells, natural killer cells; PER2, period circadian regulator 2; RORα/γ, retinoic acid receptor-related orphan receptor alpha/gamma; TME, tumor microenvironment; Tregs, regulatory T cells; VEGF, vascular endothelial growth factor.

  • Circadian clock-specific TME that affect invasive edge. Schematic illustration of the context-dependent roles of circadian clock components in regulating tumor invasion at the invasive edge. (1) EMT initiation: At the early stage of tumor invasion, core circadian regulators, including BMAL1, CLOCK, and PER2, participate in EMT initiation. This process is associated with increased MMP2/9 expression, altered F-actin dynamics, and regulation of EMT-related transcription factors (TWIST1 and SLUG), together with TGF-β-related signaling, thereby facilitating the transition from epithelial-to-mesenchymal phenotypes. (2) Invasive edge formation: At the invasive edge, circadian regulation is associated with the CSC-like states and collective invasive behavior. The CLOCK/BMAL1 axis has been implicated in promoting invasive phenotypes through downstream signaling modules (e.g., OLFML3–LGMN–CD162), whereas negative-feedback clock components, such as REV-ERBs and PER2 are associated with the modulation of stemness-related properties. External circadian perturbation, such as CJL, may further influence tumor cell behavior at the invasive edge. BMAL1, brain and muscle ARNT-like protein 1; CLOCK, circadian locomotor output cycles kaput; CJL, chronic jet lag; EMT, epithelial–mesenchymal transition; MMP2/9, matrix metalloproteinase 2/9; PER2, period circadian regulators 2; REV-ERBs, nuclear receptor subfamily 1 group D; SLUG, snail family transcriptional repressor 2; TGFβ, transforming growth factor beta; TWIST1, twist family bHLH transcription factor 1.
    Figure 3

    Circadian clock-specific TME that affect invasive edge. Schematic illustration of the context-dependent roles of circadian clock components in regulating tumor invasion at the invasive edge. (1) EMT initiation: At the early stage of tumor invasion, core circadian regulators, including BMAL1, CLOCK, and PER2, participate in EMT initiation. This process is associated with increased MMP2/9 expression, altered F-actin dynamics, and regulation of EMT-related transcription factors (TWIST1 and SLUG), together with TGF-β-related signaling, thereby facilitating the transition from epithelial-to-mesenchymal phenotypes. (2) Invasive edge formation: At the invasive edge, circadian regulation is associated with the CSC-like states and collective invasive behavior. The CLOCK/BMAL1 axis has been implicated in promoting invasive phenotypes through downstream signaling modules (e.g., OLFML3–LGMN–CD162), whereas negative-feedback clock components, such as REV-ERBs and PER2 are associated with the modulation of stemness-related properties. External circadian perturbation, such as CJL, may further influence tumor cell behavior at the invasive edge. BMAL1, brain and muscle ARNT-like protein 1; CLOCK, circadian locomotor output cycles kaput; CJL, chronic jet lag; EMT, epithelial–mesenchymal transition; MMP2/9, matrix metalloproteinase 2/9; PER2, period circadian regulators 2; REV-ERBs, nuclear receptor subfamily 1 group D; SLUG, snail family transcriptional repressor 2; TGFβ, transforming growth factor beta; TWIST1, twist family bHLH transcription factor 1.

  • Circadian clock-specific TME that affect intravasation, circulation, and extravasation. Schematic illustration of how circadian clock components regulate key steps of tumor cell dissemination, including intravasation, circulation, and extravasation, through interactions with the TME, exosome-mediated communication, and CRD. (A) Intravasation. ① The CLOCK/BMAL1 heterodimer activates the expression of NRF2, thus inhibiting secretion of IL-1β in macrophage. The CLOCK/BMAL1 heterodimer binds to the E-box to maintain the mitochondrial metabolism of macrophage homeostasis. ② RORα induces the expression of CCL2, while REV-ERBα inhibits CCL2 in macrophages. ③ CRD promotes the transition of M1 macrophages to M2 macrophages. (B) Circulation. During circulation, disseminated tumor cells are exposed to a dynamic vascular environment under conditions of circadian disruption. Circadian clock components, including BMAL1, PER2, and REV-ERBα, are associated with the regulation of tumor cell interactions with endothelial cells, platelets, and immune cells within the bloodstream, which together influence tumor cell survival and behavior during circulation. (C) Extravasation. Circadian clock genes, including CLOCK, BMAL1, and PER2, regulate endothelial activation and barrier function during extravasation. Rhythmic modulation of adhesion molecules, such as ICAM-1, together with NO signaling, influences tumor cell arrest and transmigration across the endothelium. SD and CRD exacerbate endothelial dysfunction, facilitating tumor cell extravasation into distant tissues. BMAL1, brain and muscle ARNT-like protein 1; CAF, cancer-associated fibroblast; CLOCK, circadian locomotor output cycles kaput; CRD, circadian rhythm disruption; CSCs, cancer stem cells; CCL2, C-C motif chemokine ligand 2; ECs, endothelial cells; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1 beta; NO, nitric oxide; NRF2, nuclear factor erythroid 2-related factor 2; PER1/2, period circadian regulators 1/2; RAB27A, RAB27A member RAS oncogene family; REV-ERBα, nuclear receptor subfamily 1 group D member 1; RORs, retinoic acid receptor-related orphan receptors; SD, sleep disruption; TME, tumor microenvironment.
    Figure 4

    Circadian clock-specific TME that affect intravasation, circulation, and extravasation. Schematic illustration of how circadian clock components regulate key steps of tumor cell dissemination, including intravasation, circulation, and extravasation, through interactions with the TME, exosome-mediated communication, and CRD. (A) Intravasation. ① The CLOCK/BMAL1 heterodimer activates the expression of NRF2, thus inhibiting secretion of IL-1β in macrophage. The CLOCK/BMAL1 heterodimer binds to the E-box to maintain the mitochondrial metabolism of macrophage homeostasis. ② RORα induces the expression of CCL2, while REV-ERBα inhibits CCL2 in macrophages. ③ CRD promotes the transition of M1 macrophages to M2 macrophages. (B) Circulation. During circulation, disseminated tumor cells are exposed to a dynamic vascular environment under conditions of circadian disruption. Circadian clock components, including BMAL1, PER2, and REV-ERBα, are associated with the regulation of tumor cell interactions with endothelial cells, platelets, and immune cells within the bloodstream, which together influence tumor cell survival and behavior during circulation. (C) Extravasation. Circadian clock genes, including CLOCK, BMAL1, and PER2, regulate endothelial activation and barrier function during extravasation. Rhythmic modulation of adhesion molecules, such as ICAM-1, together with NO signaling, influences tumor cell arrest and transmigration across the endothelium. SD and CRD exacerbate endothelial dysfunction, facilitating tumor cell extravasation into distant tissues. BMAL1, brain and muscle ARNT-like protein 1; CAF, cancer-associated fibroblast; CLOCK, circadian locomotor output cycles kaput; CRD, circadian rhythm disruption; CSCs, cancer stem cells; CCL2, C-C motif chemokine ligand 2; ECs, endothelial cells; ICAM-1, intercellular adhesion molecule 1; IL-1β, interleukin-1 beta; NO, nitric oxide; NRF2, nuclear factor erythroid 2-related factor 2; PER1/2, period circadian regulators 1/2; RAB27A, RAB27A member RAS oncogene family; REV-ERBα, nuclear receptor subfamily 1 group D member 1; RORs, retinoic acid receptor-related orphan receptors; SD, sleep disruption; TME, tumor microenvironment.

  • Circadian clock cell-cycle crosstalk on tumor metastasis. Schematic illustration of the interactions between circadian clock components and cell-cycle regulators controlling transitions through G1, S, G2, and M phases. G1 phase: Cell-cycle entry is governed by cyclin D–Cdk4/6. Under CCD, cyclin D–Cdk4/6 activity is modulated with PER2 shown as an inhibitory regulator. p21/p27 act as checkpoint mediators at the G1/S boundary and are influenced by PER1, REV-ERBα, RORα, and CJL. These interactions collectively regulate progression toward the S phase. S phase: Progression through S phase involves cyclin E–Cdk2 and cyclin A–Cdk2 complexes. PER1 is linked to cyclin E–Cdk2 regulation, while REV-ERBα modulates cyclin A–Cdk2 activity, contributing to S-phase control. G2 phase: Transition into mitosis is coordinated by cyclin B–Cdk1. BMAL1/CLOCK regulate WEE1, which in turn modulates cyclin B–Cdk1 activity. CRY1 and PER1 influence WEE1, further integrating circadian control at the G2 checkpoint. M phase and G2/M checkpoint: Circadian components intersect with DDR pathways, including PER2–CHK2, CRY1–TIM–ATR–CHK1, and PER1–TIM–ATM–CHK2, which modulate cyclin B–Cdk1-dependent mitotic progression. External cues, such as DD are depicted as additional modulators at this stage. ATR, ATR serine/threonine kinase; ATM, ATM serine/threonine kinase; BMAL1, brain and muscle ARNT-like protein1; CCD, chronic circadian disruption; CDK, cyclin-dependent kinase; CHK1/2, checkpoint kinase1/2; Cdk1/2/4/6, cyclin dependent kinase 1/2/4/6; CLOCK, circadian locomotor output cycles kaput; CRY1, cryptochrome1; CJL, chronic jet-lag; DD, constant dark; PER1/2, period circadian regulator1/2; RORα, retinoic acid receptor-related orphan receptorα; REV-ERBα, nuclear receptor subfamily 1 group d member; TIM, timeless; WEE1, G2 checkpoint kinase.
    Figure 5

    Circadian clock cell-cycle crosstalk on tumor metastasis. Schematic illustration of the interactions between circadian clock components and cell-cycle regulators controlling transitions through G1, S, G2, and M phases. G1 phase: Cell-cycle entry is governed by cyclin D–Cdk4/6. Under CCD, cyclin D–Cdk4/6 activity is modulated with PER2 shown as an inhibitory regulator. p21/p27 act as checkpoint mediators at the G1/S boundary and are influenced by PER1, REV-ERBα, RORα, and CJL. These interactions collectively regulate progression toward the S phase. S phase: Progression through S phase involves cyclin E–Cdk2 and cyclin A–Cdk2 complexes. PER1 is linked to cyclin E–Cdk2 regulation, while REV-ERBα modulates cyclin A–Cdk2 activity, contributing to S-phase control. G2 phase: Transition into mitosis is coordinated by cyclin B–Cdk1. BMAL1/CLOCK regulate WEE1, which in turn modulates cyclin B–Cdk1 activity. CRY1 and PER1 influence WEE1, further integrating circadian control at the G2 checkpoint. M phase and G2/M checkpoint: Circadian components intersect with DDR pathways, including PER2–CHK2, CRY1–TIM–ATR–CHK1, and PER1–TIM–ATM–CHK2, which modulate cyclin B–Cdk1-dependent mitotic progression. External cues, such as DD are depicted as additional modulators at this stage. ATR, ATR serine/threonine kinase; ATM, ATM serine/threonine kinase; BMAL1, brain and muscle ARNT-like protein1; CCD, chronic circadian disruption; CDK, cyclin-dependent kinase; CHK1/2, checkpoint kinase1/2; Cdk1/2/4/6, cyclin dependent kinase 1/2/4/6; CLOCK, circadian locomotor output cycles kaput; CRY1, cryptochrome1; CJL, chronic jet-lag; DD, constant dark; PER1/2, period circadian regulator1/2; RORα, retinoic acid receptor-related orphan receptorα; REV-ERBα, nuclear receptor subfamily 1 group d member; TIM, timeless; WEE1, G2 checkpoint kinase.

  • Chronobiology reporting standards in circadian research. Schematic overview of key methodologic parameters that should be reported in circadian studies, including Zeitgeber time definition, light–dark cycle conditions, gender and age of experimental subjects, jet lag or chronic circadian disruption paradigms, and time-aware statistical methods.
    Figure 6

    Chronobiology reporting standards in circadian research. Schematic overview of key methodologic parameters that should be reported in circadian studies, including Zeitgeber time definition, light–dark cycle conditions, gender and age of experimental subjects, jet lag or chronic circadian disruption paradigms, and time-aware statistical methods.

Tables

  • Table 1

    CLOCK gene expression across various cancer types

    NameCancer typeModelTypeMetastatic endpointEffect on cancerReferences
    BMAL1CRCHuman tissuemRNALiver metastasisIncreased metastasis of liver24
    CRCMouse modelProtein-Longer OS112
    Nasopharyngeal carcinomaHuman tissueProteinDistant metastasisDecreased tumor distant metastasis26
    BMAL2CRCHuman tissuemRNALiver metastasisIncreased metastasis of liver25
    TIMELESSBreast cancerHuman tissue/cell linesmRNA-Shorter MFS22
    Cervical cancerHuman tissueProteinPelvic lymph node metastasisIncreased metastasis of pelvic lymph nodes, shorter DFS113
    PER1Breast cancerHuman tissue/cell linesmRNA-Longer MFS22
    CRCHuman tissuemRNALiver metastasisDecreased metastasis of liver24
    PER2Breast cancerHuman tissueProteinLymph node metastasisIncreased metastasis of lymph nodes21
    Breast cancerHuman tissue/cell linesmRNA-Longer MFS22
    PER3Breast cancerHuman tissue/cell linesmRNA-Longer MFS22

    CRC
    mRNA
    Liver metastasis
    Decreased metastasis of liver    		114,115
    CRY2Breast cancerHuman tissue/cell linesmRNA-Longer MFS22
    REV-ERBαGastric cancerHuman tissueProtein-Decreased metastasis of tumor and lymph nodes28
    RORCBreast cancerHuman tissue/cell linesmRNA-Longer MFS22
    NPAS2Breast cancerHuman tissue/cell linesmRNALonger MFS22
    HLFNSCLCHuman tissue/xenograft modelmRNA/proteinDistant metastasisDecreased tumor distant metastasis29

    OS, overall survival; DFS, disease-free survival; MFS, metastasis-free survival; CRC, colorectal cancer; NSCLC, non-small cell lung cancer.

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