Circadian Clock Genes in Colorectal Cancer: From Molecular Mechanisms to Chronotherapeutic Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes the role of circadian rythm-associated molecules in a colorectal cancer development. The topic has scientific value and the paper represents novel data about chronobiology and cancer.

Author Response

Comments 1: The manuscript describes the role of circadian rythm-associated molecules in a colorectal cancer development. The topic has scientific value and the paper represents novel data about chronobiology and cancer.

Response 1: Sincerely thank you for devoting your precious time to reviewing this manuscript and providing your recognition! Taking into account your valuable comments as well as those of other reviewers, we have made systematic revisions to multiple aspects of the manuscript's logical structure and expression standards, aiming to further enhance its readability and academic value.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the review article titled “Circadian Clock Genes in Colorectal Cancer: From Molecular Mechanisms to Chronotherapeutic Applications” Gangping Li and co-authors describe advances and perspectives of exploring the role of the circadian clock circuitry in colorectal cancer.

 

The paper contains some new information.

The review design is clear and sound.

There are no apparent errors of fact or logic.

The discussion and conclusions are well balanced and adequately supported by the data.

 

There are few typos. I suggest corrections and I recommend accurate proofreading of the manuscript

 

 

Author Response

Comments 1: In the review article titled “Circadian Clock Genes in Colorectal Cancer: From Molecular Mechanisms to Chronotherapeutic Applications” Gangping Li and co-authors describe advances and perspectives of exploring the role of the circadian clock circuitry in colorectal cancer.

Response 1: Thank you for your valuable summary! We have further supplemented content related to potential translational entry points for the future, aiming to further enhance the logical coherence and academic value of the manuscript.

Comments 2: The paper contains some new information.

Response 2: We are sincerely delighted with your recognition and affirmation of this review! Compared with currently published literature related to clock genes and CRC, this review has conducted a more systematic collation and integration of relevant research findings from the dual perspectives of multiple pathogenic mechanisms and clinical therapeutic applications. Furthermore, in this revision, we have further supplemented translational medicine entry points with potential application value, aiming to further enhance the innovation and academic value of this review.

Comments 3: The review design is clear and sound.

Response 3: As you have noted, at the basic research level, this review systematically clarifies the specific regulatory roles of different clock genes in the pathological progression of CRC by focusing on multiple pathogenic mechanisms; at the clinical application level, it comprehensively summarizes the application value and underlying mechanisms of identified clock genes in CRC diagnosis and treatment, approached from the dual dimensions of diagnosis and therapy.

Comments 4: There are no apparent errors of fact or logic.

Response 4: Sincerely thank you for your valuable recognition of this review!

Comments 5: The discussion and conclusions are well balanced and adequately supported by the data.

Response 5: Sincerely thank you for your valuable recognition of this review! Besides, taking into account your valuable comments as well as those of many other reviewers, we have made targeted revisions to the conclusions with ambiguous expressions and related mechanisms of action at multiple places in the manuscript, aiming to provide readers with a clearer and more coherent reading experience.

Comments 6: There are few typos. I suggest corrections and I recommend accurate proofreading of the manuscript.

Response 6: Thank you for your valuable and meticulous reading! We have addressed the several grammatical errors you identified and corrected each one, and we have also conducted a second thorough review and check of the entire revised manuscript to ensure that there are no other overlooked errors. For example on Page 4, Line 116 “which may result in poorer outcome” and so on.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this review, Wang and colleagues examine the role of circadian clock genes in colorectal cancer. The manuscript offers an up-to-date and wide-ranging synthesis of molecular mechanisms, metabolic regulation, microenvironmental influences, and potential clinical applications, including chronotherapy. The topic is highly relevant, and the review has clear strengths, particularly the breadth of literature covered and the integration of basic and translational perspectives.
However, the manuscript would benefit from substantial improvements in organization, clarity, and precision. Below I provide both general comments and specific suggestions aimed at supporting a clearer and more impactful final version.

General Comments

• Across the manuscript, many sentences are too long and dense, which makes readability difficult. Breaking them into shorter, clearer statements would markedly improve the flow.
• Several concepts are repeated multiple times across different sections (e.g., variability between animal models, lack of standardized chronotherapy frameworks, small sample sizes, and the need for omics-based approaches). Consolidating these ideas into a single, well-structured paragraph or section would increase conciseness and avoid redundancy.
• The manuscript moves frequently between observations in zebrafish, murine models, Drosophila, human tissues, and cancer cell lines. In many places, the relevance or translational limitations of each model are not clearly articulated. Please explicitly identify the model system in each instance and briefly contextualize its limitations.
• The review is descriptive in many parts. A deeper critical appraisal—for example, when contradictory findings are presented—would enhance the manuscript’s value.
• The manuscript lists numerous mechanisms involving BMAL1, CLOCK, PER, CRY, TIM, CK1ε, and REV-ERBs. However, it does not clearly articulate which pathways represent primary drivers versus secondary consequences. For example: Is the dominant circadian influence on CRC mediated through the cell cycle, metabolism, or the immune microenvironment? Are BMAL1 and CLOCK upstream regulators of most observed effects, or do these pathways operate in parallel? A clearer mechanistic hierarchy would help synthesize the field.
• CRC is a highly heterogeneous disease, but this review only briefly mentions right- and left-sided tumor differences (Lines 332–334). Please add a dedicated paragraph discussing how CRC molecular subtypes may modulate circadian gene expression or responses to circadian-based interventions.
• The circadian–microbiome–immune axis requires deeper discussion. For example: does dysbiosis independently disrupt local intestinal clocks (epithelial or immune cells)? Which microbial metabolites show rhythmicity, and are these altered in CRC? Could microbiome-based interventions (e.g., time-restricted feeding, pre/probiotics) synergize with chronotherapy?
• The review reports multiple immune pathways influenced by circadian disruption but does not fully explain which immune populations show intrinsic circadian oscillations, how local versus systemic circadian disruption differ, or which cell types drive the observed microenvironmental changes. A more structured system-level discussion would enhance clarity.
• Clock gene expression changes are presented primarily as transcriptional or functional modifications, but epigenetic regulation is a major emerging field that should be integrated into the discussion.
• The impact of circadian rhythms on chemotherapy resistance is only briefly addressed. This is a key translational aspect and deserves more depth, particularly regarding drug metabolism, DNA repair rhythms, and pharmacokinetics.
• There is no discussion of how CRC itself may disrupt host circadian rhythms (e.g., via cytokine production, altered feeding/activity patterns, or metabolic reprogramming). Including this “bidirectional disruption” perspective would enrich the review.
• Please revise the citation formatting to ensure that reference numbers are placed before the final punctuation mark of the sentence they refer to, rather than after it.

Specific Comments

• Lines 101–104: Please correct the sentence “…which may resulting in poorer outcome.” to “…which may result in poorer outcomes.”
• Lines 161–166: The BMAL1 versus CLOCK influences are compressed into one sentence. I suggest dividing this into two or three sentences and providing more detail on the distinct mechanisms involved.
• Lines 171–175: Please provide a brief description of how BMAL1 influences exosome secretion to strengthen this point; otherwise, the reference to exosomes appears disconnected from the surrounding discussion.
• Lines 197–216: This paragraph is overly dense, covering glycolysis, immunity, metastasis, and methodological recommendations. Splitting it into 2–3 thematic paragraphs would greatly improve clarity.
• Lines 218–220: This paragraph requires clearer explanation and transitions. Please clarify the mechanistic flow.
• Lines 327–354: Statements such as “may obtain multiple types of information” are vague. Please specify which markers show the strongest diagnostic or prognostic promise.
• Conclusions: Several limitations repeat earlier statements. This section would benefit from being condensed and structured around: (1) established mechanisms; (2) emerging connections; (3) clear research priorities; and (4) translational challenges and opportunities. This would make the conclusion more impactful.

Figures and Tables

• Figure 1: The caption should more clearly explain the interactions between environmental cues and the TTFL system.
• Figure 2: Although an effective visual summary, please consider referencing it explicitly in the clinical sections for greater integration.
• Tables 1 and 2: Some fields use “unmentioned” and “not mentioned” inconsistently. Please standardize terminology.

Comments on the Quality of English Language

The English is generally understandable but contains grammatical inconsistencies, ambiguous phrasing, and stylistic issues. For example, replace “deregulation” with “dysregulation”, and “secondary to” with “leading to” or “resulting in”. Several paragraphs would benefit from stylistic revision to reduce complexity and improve clarity.

Author Response

Comments 1: Across the manuscript, many sentences are too long and dense, which makes readability difficult. Breaking them into shorter, clearer statements would markedly improve the flow.

Response 1: We sincerely appreciate your valuable suggestions! In the previous manuscript, we did use a considerable number of subordinate clauses, which has resulted in many sentences requiring readers to sort out the subject of description during reading. In accordance with your suggestions, we have revised many expressions, breaking down the original subordinate clause structures into multiple short sentences with the aim of providing readers with a smoother reading experience. For examples, on Page 3, Line 87-105 ”The Wnt pathway serves as a key intercellular coupling component linking intestinal circadian rhythms to the cell cycle. However, the tumor suppressor effect of PER2 on the Wnt pathway varies across different animal models. In in vitro cell models and mouse models, PER2 has been identified as a tumor suppressor gene, as it maintains the normal expression rhythms of c-Myc and Cyclin D [24]; in contrast, PER2 deficiency in zebrafish xenograft models and PER2 knockout in Drosophila do not induce carcinogenesis [25,26]. This discrepancy may be attributed to two factors: first, the presence of other PER family isoforms in zebrafish and Drosophila which compensate for the cyclin-related regulatory function of human PER2 (e.g., BmPER); second, the relatively complex PER2-related signaling pathways in mammals. This also suggests that for studies exploring the role of clock genes in human diseases, mammalian models are preferred to ensure the consistency of gene function. Notably, PER2 exhibits relatively consistent functions in maintaining the stability of the cell proliferation cycle.”
Page 4, Line 117-125 “Additionally, BMAL1 maintains the normal self-renewal balance of intestinal stem cells (ISCs) by inhibiting excessive activation of the Hippo signaling pathway, e.g., regulating Yap nuclear localization activity. Its depletion induces abnormal activation of the Hippo pathway, accompanied by downregulation of the Wnt pathway, and subsequently triggers cell cycle dysregulation and abnormal excessive proliferation of ISCs [29]. Although other studies have reported contradictory effects of BMAL1 on ISC proliferation [30], this discrepancy may stem from differences in study model conditions, such as non-cancer context, and ISC subpopulation focus. Nevertheless, all these studies highlight the indispensable core role of BMAL1 in maintaining ISC proliferation homeostasis.”
Page 6, Line 230-238 ”As mentioned earlier, the activation of the Wnt signaling pathway is mediated by BMAL1 deletion. The additional enhancement of glycolysis is driven by the subsequent upregulation of c-Myc downstream. Both of these effects have been validated in mouse intestinal organoids and CRC patient-derived organoids. The TCGA-COAD database further confirms that CRC patients with this molecular signature (aberrant activation of the Wnt-c-Myc-glycolysis axis) have significantly shorter overall survival (Log-rank P=0.0032) [50]. Based on the aforementioned experimental and clinical evidence, the regulation of the Wnt pathway via clock genes may be the core pathological pathway during the occurrence and development of CRC.”
And so on.

Comments 2: Several concepts are repeated multiple times across different sections (e.g., variability between animal models, lack of standardized chronotherapy frameworks, small sample sizes, and the need for omics-based approaches). Consolidating these ideas into a single, well-structured paragraph or section would increase conciseness and avoid redundancy.

Response 2: We sincerely appreciate your valuable suggestions! Your feedback that some viewpoints regarding existing deficiencies are repetitive is highly pertinent. To balance providing readers with sufficient information and ensuring reading fluency, in accordance with your suggestions, we have deleted redundant content related to existing technical barriers, retaining only the most core deficiencies. Meanwhile, in the Conclusion section, we have briefly echoed the existing issues mentioned earlier and further extended and deepened the discussion. For example, regarding the issue of animal model selection for cell cycle-related research mentioned on Page 3, Lines 96-99, “This also suggests that for studies exploring the role of clock genes in human diseases, mammalian models are preferred to ensure the consistency of gene function. Notably, PER2 exhibits relatively consistent functions in maintaining the stability of the cell proliferation cycle” we have edited the redundant repetition of this content at the end of this section on Page 4, Line 129-142. We have also supplemented the explanation of the potential causes leading to these conclusion differences in the original manuscript and corroborated them with specific cases. Furthermore, on Page 15, Lines 533-536, ”At the animal model level, mammalian models should be prioritized, and the differences in the active periods of the biological clock between model animals and humans must be strictly corrected to ensure the clinical applicability of research results” we have briefly echoed the relevant key points of animal model selection mentioned earlier and made appropriate extensions and supplements.

Comments 3: The manuscript moves frequently between observations in zebrafish, murine models, Drosophila, human tissues, and cancer cell lines. In many places, the relevance or translational limitations of each model are not clearly articulated. Please explicitly identify the model system in each instance and briefly contextualize its limitations.

Response 3: Your suggestions are of great guiding value! Your observation that the cited literature involves a variety of animal models in the relevant discussion sections of cell cycle and tumor metastasis is highly pertinent. In accordance with your suggestions, we have supplemented the corresponding animal model descriptions at the locations of the relevant literature citations; for the divergent conclusions drawn regarding the same issue across different animal models, we have further added possible mechanistic explanations and put forward corresponding research improvement suggestions on Page 3, Line 87-105 “The Wnt pathway serves as a key intercellular coupling component linking intestinal circadian rhythms to the cell cycle. However, the tumor suppressor effect of PER2 on the Wnt pathway varies across different animal models. In in vitro cell models and mouse models, PER2 has been identified as a tumor suppressor gene, as it maintains the normal expression rhythms of c-Myc and Cyclin D [24]; in contrast, PER2 deficiency in zebrafish xenograft models and PER2 knockout in Drosophila do not induce carcinogenesis [25,26]. This discrepancy may be attributed to two factors: first, the presence of other PER family isoforms in zebrafish and Drosophila which compensate for the cyclin-related regulatory function of human PER2 (e.g., BmPER); second, the relatively complex PER2-related signaling pathways in mammals. This also suggests that for studies exploring the role of clock genes in human diseases, mammalian models are preferred to ensure the consistency of gene function. Notably, PER2 exhibits relatively consistent functions in maintaining the stability of the cell proliferation cycle.
TIM functions as an oncogene, and is highly expressed during the S and G2 phases of DNA replication and cell division. Its depletion induces G2-phase cell cycle arrest in human colorectal cancer cell lines (e.g., HCT116). Notably, widespread TIM overexpression is observed in clinical colorectal cancer specimens. These observations are closely associated with TIM’s involvement in regulating the phosphorylation of key cell cycle kinases, including CDK1. Additionally, TIM may also function to maintain genomic stability [27]” and on Page 5, Line 164-179 ”TIM inhibits CRC metastasis. Multiple clinical studies confirm that TIM expression level is negatively correlated with CRC metastatic potential. In the zebrafish xenotransplantation model, TIM knockdown significantly enhances the in vivo metastatic potential of CRC cells. The nude mouse model further uncovered the molecular mechanism underlying this phenomenon: TIM depletion reduces the expression levels of epithelial markers (e.g., E-cadherin/CDH1) significantly, while upregulating the expression of mesenchymal markers (e.g., Vimentin, FN1) and the EMT core transcription factor ZEB1. This thereby drives the EMT process and ultimately enhances the invasive ability of CRC cells [39]. Although research on ZEB1-targeted immunotherapeutic strategies for CRC has been reported, ZEB1 expression is regulated by multiple upstream factors (e.g., transcription factors like SNAIL), and this may increase the complexity of targeted intervention. Thus, developing targeted strategies for TIM, the upstream regulator of ZEB1, may be a more promising direction. Compared with ZEB1, TIM has a simpler expression regulatory network, is less disturbed by other irrelevant factors, and can more precisely affect the immune microenvironment and malignant phenotype of CRC by regulating the TIM-ZEB1 axis [40].”

Comments 4: The review is descriptive in many parts. A deeper critical appraisal—for example, when contradictory findings are presented—would enhance the manuscript’s value.

Response 4: We agree with your concept. In accordance with your suggestions, for the contradictory conclusions emerging in the research—especially the outcome differences induced by different animal models—we have conducted supplementary searches of relevant literature. We have also supplemented the explanation of the potential causes leading to these conclusion differences in the original manuscript and corroborated them with specific cases on Page 3, Line 87-99 ”The Wnt pathway serves as a key intercellular coupling component linking intestinal circadian rhythms to the cell cycle. However, the tumor suppressor effect of PER2 on the Wnt pathway varies across different animal models. In in vitro cell models and mouse models, PER2 has been identified as a tumor suppressor gene, as it maintains the normal expression rhythms of c-Myc and Cyclin D [24]; in contrast, PER2 deficiency in zebrafish xenograft models and PER2 knockout in Drosophila do not induce carcinogenesis [25,26]. This discrepancy may be attributed to two factors: first, the presence of other PER family isoforms in zebrafish and Drosophila which compensate for the cyclin-related regulatory function of human PER2 (e.g., BmPER); second, the relatively complex PER2-related signaling pathways in mammals. This also suggests that for studies exploring the role of clock genes in human diseases, mammalian models are preferred to ensure the consistency of gene function. Notably, PER2 exhibits relatively consistent functions in maintaining the stability of the cell proliferation cycle” and on Page 4, Line 122-125, ”Although other studies have reported contradictory effects of BMAL1 on ISC proliferation [30], this discrepancy may stem from differences in study model conditions, such as non-cancer context, and ISC subpopulation focus. Nevertheless, all these studies highlight the indispensable core role of BMAL1 in maintaining ISC proliferation homeostasis”, Line 129-142 “Overall, the regulatory role of clock genes in the cell cycle of CRC has been partially elucidated. Through multiple signaling pathways such as Wnt and key molecules in-cluding p53 and Wee1, they collectively maintain cell cycle homeostasis. Dysregulated expression of clock genes thus leads to cell cycle dysregulation, which in turn promotes CRC tumorigenesis and progression. Currently, further exploration of the underlying mechanisms in this field still faces several challenges. First, although the core function of clock genes in stabilizing the cell cycle is generally conserved across non-mammalian models, there may be significant differences in the details of regulatory mechanisms. Therefore, if non-mammalian models yield conclusions inconsistent with those from mammalian models, research on this pathological mechanism should primarily take the results of mammalian models as the core reference. Second, research on some members of the clock gene family remains largely incomplete. Existing evidence indicates strong correlations between the regulatory mechanisms of different clock genes. Filling these research gaps will therefore provide important support for the development of this field.” 

Comments 5: The manuscript lists numerous mechanisms involving BMAL1, CLOCK, PER, CRY, TIM, CK1ε, and REV-ERBs. However, it does not clearly articulate which pathways represent primary drivers versus secondary consequences. For example: Is the dominant circadian influence on CRC mediated through the cell cycle, metabolism, or the immune microenvironment? Are BMAL1 and CLOCK upstream regulators of most observed effects, or do these pathways operate in parallel? A clearer mechanistic hierarchy would help synthesize the field.

Response 5: We sincerely appreciate you raising this critical question! The regulation of various pathological processes in CRC by clock genes is not dominated by a single gene, but rather by the synergistic effect of dysregulated expression of multiple clock genes. Meanwhile, clock genes form a TTFL, and their respective expression levels exhibit mutual regulatory relationships that may induce chain reactions. In addition, the collection time of pathological tissues is difficult to standardize, and it remains undetermined which clock gene is the first to exhibit dysregulated expression.
Notably, there are key regulatory pathways involved in many pathological processes of CRC: the downstream target genes of multiple clock genes converge on these pathways to a certain extent. For example, the Wnt pathway mentioned in this manuscript plays a central role in the regulation of the CRC, like on Page 6, Line 236-238 “Based on the aforementioned experimental and clinical evidence, the regulation of the Wnt pathway via clock genes may be the core pathological pathway during the occurrence and development of CRC.”

Comments 6: CRC is a highly heterogeneous disease, but this review only briefly mentions right- and left-sided tumor differences (Lines 332–334). Please add a dedicated paragraph discussing how CRC molecular subtypes may modulate circadian gene expression or responses to circadian-based interventions.

Response 6: Your perspective is of great reference value! Considering that CRC has multiple pathological subtypes and the microbiota variability at different colorectal locations is significant, the heterogeneity of gene expression in its tissues may be more prominent. Thus, we only briefly mention this part, and its underlying cause is likely related to microbiota heterogeneity. However, this heterogeneity has not received sufficient attention in existing mechanistic studies—on the one hand, because the same dysregulated expression of clock genes occurs frequently across multiple subtypes; on the other hand, the focus of existing clinical studies is mostly on the other factors such as gender. Therefore, we believe that the available reference data on this issue are currently insufficient, and it is not yet appropriate to devote a separate paragraph to elaborate on it.

Comments 7: The circadian–microbiome–immune axis requires deeper discussion. For example: does dysbiosis independently disrupt local intestinal clocks (epithelial or immune cells)? Which microbial metabolites show rhythmicity, and are these altered in CRC? Could microbiome-based interventions (e.g., time-restricted feeding, pre/probiotics) synergize with chronotherapy?

Response 7: We sincerely appreciate your valuable suggestions, and we are delighted to note your interest in this section! In accordance with your comments, we have comprehensively optimized the content and expression of this paragraph, and accurately addressed the two core questions you raised: "whether gut microbiota dysbiosis can independently disrupt local intestinal clocks" and "which microbial metabolites exhibit rhythmicity and their alterations in CRC" on Page 8, Line 324-367 “Although significant progress has been made in research on the regulatory mechanisms of non-intestinal-specific immune cells, studies on the CRC microenvironment need to incorporate a key factor unique to the intestinal system: the role of the gut microbiota [71]. Dysregulation of the gut microbiota and its metabolites (e.g., taurocholic acid (TCA)) interacts with the circadian rhythm to trigger inflammatory responses, and microenvironmental changes may be an important link in the pathogenic process of CRC, thereby affecting CRC progression, treatment response, tumor metastasis, and clinical prognosis [72,73].
Notably, gut microbiota dysbiosis can independently disrupt local intestinal clocks, and many of its derived metabolites inherently exhibit distinct circadian rhythms are significantly dysregulated in CRC. Under physiological conditions, the levels of short-chain fatty acids (SCFAs) oscillate circadianly, peaking during the dark phase in mice, and it is synchronized with the rhythmic expression of intestinal clock genes (e.g., BMAL1, Per2); as a secondary bile acid metabolized by the microbiota, TCA’s diurnal fluctuations are also tightly coupled to the intestinal circadian rhythm [72]. In contrast, feeding during the rest period (a factor that directly induces microbiota dysbiosis) can alter the phase of the colonic peripheral circadian clock and cause microbiota dysbiosis in mice, reducing SCFA-producing bacteria and butyrate levels, which in turn decreases Treg cell density, leads to increased intestinal barrier permeability, and promotes CRC development [6]. This indicates that changes in microbiota composition can independently affect the clock rhythm of intestinal epithelial cells through metabolite mediation, without relying on central clock regulation. Meanwhile, CRC-related microbiota dysbiosis (e.g., reduced abundance of Bifidobacterium and Lactobacillus) can disrupt the intrinsic circadian oscillations of intestinal immune cells (e.g., macrophages); for example, decreased butyrate impairs the rhythmic expression of BMAL1 and REV-ERBα in colonic macrophages, triggering sustained inflammation, which echoes the core mechanism of REV-ERBα regulating macrophage inflammation [68]. These changes not only decouple microbial metabolites from the host circadian rhythm but also exacerbate pathological processes by regulating immune cell function: For instance, TCA can epigenetically promote glycolysis in MDSCs, enhance the monomethylation of the target gene H3K4, and inhibit CHIP-mediated ubiquitylation of PDL1, leading to the aggregation of MDSCs and dysfunctional CD8+ T cells in the lungs of mice. This weakens tumor-specific immune function and promotes CRC progression and lung metastasis [61,73].
Overall, gut microbial oscillations may be an important factor associated with the host circadian rhythm and tumor microenvironment, and there are both interacting links and independent regulatory components between the gut microbiota and circadian rhythms. However, microbiome science is still in its early stages of development, and its application in CRC faces numerous challenges: the lack of longitudinal population cohorts with stool samples and sufficient clinical metadata to support relevant analyses, the need for in-depth exploration of the causal relationships between the microbiota, its metabolites, and various pathological processes, as well as the significant impact of experimental factors such as inter-individual heterogeneity in microbiota composition and sampling time on research reproducibility [71]. These are all long-term issues that need to be ad-dressed in this field in the future.”
Regarding the last question you mentioned (whether microbiome-based interventions can synergize with chronotherapy), considering that the definition of chronotherapy has not been introduced in this paragraph, we have supplemented and elaborated on the detailed discussion of the relevant synergistic mechanisms in subsequent sections to ensure the coherence of the discussion logic on Page 16, Line 518-528 Additionally, there are two other research directions that have been relatively less explored but still hold great translational potential. Based on the potential synergy between gut microbiota-based interventions and chronotherapy mentioned earlier, this combined approach can be more easily translated into clinical practice. Time-restricted feeding (TRF) can reshape colonic circadian rhythms by regulating gut microbiota composition (e.g., restoring SCFA-producing bacteria) [6]. Supplementing SCFA-producing probiotics (e.g., Bifidobacterium breve) or prebiotics (e.g., inulin) can restore the circadian rhythms of microbial metabolites and intestinal clocks. When combined with chronotherapy, ad-ministering REV-ERBα agonists during peak inflammatory periods can synergistically inhibit NLRP3-mediated inflammation and further enhance anti-tumor immunity [68].”

Comments 8: The review reports multiple immune pathways influenced by circadian disruption but does not fully explain which immune populations show intrinsic circadian oscillations, how local versus systemic circadian disruption differ, or which cell types drive the observed microenvironmental changes. A more structured system-level discussion would enhance clarity.

Response 8: We sincerely appreciate your valuable suggestions! Considering that the manuscript has consistently adopted the core segmentation logic of "different clock genes and their regulatory mechanisms", we have not adjusted the overall framework to maintain structural consistency, but only optimized certain logical expressions. Nevertheless, we have addressed the questions you raised as follows:
1. Clarifying immune populations with intrinsic circadian rhythms
Macrophages: These cells possess distinct intrinsic circadian oscillators, and core clock genes such as BMAL1 and REV-ERBα can rhythmically regulate their inflammatory responses [71,72]. This manuscript emphasizes the inhibitory effect of REV-ERBα on the NLRP3 inflammasome in macrophages—this regulation has an inherent time dependence, as the expression of REV-ERBα itself exhibits a circadian oscillation pattern, thereby regulating macrophage-mediated inflammatory responses in a rhythmic manner.
T cells: Tregs exhibit intrinsic circadian rhythms in proliferation capacity and cytokine secretion [73]. The manuscript mentions that BMAL1 deletion leads to a reduction in CD4+ T cell numbers, and we further clarify here: this reduction is partly due to the disruption of the intrinsic circadian rhythm of CD4+ T cells themselves (e.g., impaired activation and survival capacity regulated by circadian rhythms) and is also indirectly influenced by Breg cells.
MDSCs: The migration and recruitment of neutrophils are clearly regulated by intrinsic circadian rhythms [74], which is consistent with the observation in this manuscript that "BMAL1 disruption enhances neutrophil migration capacity". The suppressive activity of MDSCs also exhibits circadian oscillations and is regulated by clock genes such as BMAL1 [75], providing mechanistic support for the finding of "MDSC aggregation in the TME" in this manuscript.
2. Differences between local and systemic circadian disruption
Local circadian disruption: Specifically refers to rhythm abnormalities within the TME (e.g., tumor cells, adjacent immune cells, and stromal cells). Specifically, BMAL1 deletion in CRC cells can directly upregulate the Wnt/c-Myc signaling pathway, leading to local overexpression of Cxcl5 and subsequent recruitment of neutrophils/MDSCs to the TME—this type of disruption has cell-autonomous characteristics, is confined to the local tumor microenvironment, and mainly affects the interactions between local immune cells and inflammatory responses.
Systemic circadian disruption: Originates from dysfunction of the central clock or systemic rhythm disturbances (e.g., shift work, jet lag), and regulates peripheral tissues through neuroendocrine and autonomic nervous signals [76]. Such disruption can indirectly affect CRC progression by altering systemic immune homeostasis—for example, systemic REV-ERBα rhythm disruption may comprehensively impair the anti-inflammatory function of macrophages, while systemic BMAL1 dysfunction leads to a reduction in CD4+ T cell numbers in the circulatory system (not limited to the TME). Notably, local and systemic disruptions often interact: factors such as inflammatory cytokines produced locally by tumors may further perturb systemic rhythms [77], forming a positive feedback loop that exacerbates immunosuppression.
3. Cell types driving TME changes
Tumor cells: Alterations in clock genes in CRC cells (e.g., BMAL1 knockout, REV-ERBα downregulation) drive TME changes through cell-autonomous signaling pathways—BMAL1-deficient tumor cells secrete Cxcl5 to recruit neutrophils/MDSCs, while REV-ERBα deletion enhances NLRP3-mediated inflammatory responses.
Macrophages: As core immune regulatory cells in the TME, macrophages mediate REV-ERBα/NLRP3-dependent inflammatory responses; their intrinsic circadian rhythms further regulate the timing and intensity of these responses.
Breg cells and CD4+ T cells: BMAL1 disruption leads to a decrease in IL-33 levels, which in turn reduces the number of PD-L1-expressing Breg cells and ultimately downregulates CD4+ T cells, directly impairing anti-tumor immunity within the TME.
Endothelial cells: The expression of CRY1/CRY2 in endothelial cells regulates the secretion of NF-κB and inflammatory factors; their dysfunction exacerbates vascular inflammation and promotes the recruitment of immune cells into the TME.

Comments 9: Clock gene expression changes are presented primarily as transcriptional or functional modifications, but epigenetic regulation is a major emerging field that should be integrated into the discussion.

Response 9: We sincerely appreciate your valuable suggestions! Supplementing relevant content in this field can indeed further improve our manuscript. In accordance with your suggestions, we have conducted a literature search focusing on relevant directions, including clinical cohort studies and other types, and have supplemented the relevant content into the manuscript on Page 16, Line 528-532 “Existing studies have shown that epigenetic modifications targeting clock genes (such as methylation and histone acetylation) can also exert regulatory effects on various downstream physiological processes, including metabolism and cell cycle. Therefore, subsequent studies on the epigenetic modifications of clock genes will also be one of the highly promising research avenues [101,102].”

Comments 10: The impact of circadian rhythms on chemotherapy resistance is only briefly addressed. This is a key translational aspect and deserves more depth, particularly regarding drug metabolism, DNA repair rhythms, and pharmacokinetics.

Response 10: Your focus is of great significance in translational research! However, [74] and many other relevant studies on chronotherapy combined with chemotherapy have not clearly addressed the specific changes in pharmacokinetics and the detailed impacts of related mechanisms such as DNA repair.
Currently, the core idea of chronotherapy remains to administer drugs during periods when the expression level of drug-metabolizing enzymes is low, or to regulate clock genes to increase the levels of substances related to therapeutic efficacy, thereby enhancing treatment outcomes.

Comments 11: There is no discussion of how CRC itself may disrupt host circadian rhythms (e.g., via cytokine production, altered feeding/activity patterns, or metabolic reprogramming). Including this “bidirectional disruption” perspective would enrich the review.

Response 11: Thank you for your supplementary suggestions! Regarding the impact of peripheral clocks on central clocks, we have only briefly mentioned it in Figure 1 so far—with relatively limited relevant research available, and the specific regulatory mechanisms have not yet been clarified. Existing studies suggest that this effect may be related to the regulatory role of substances such as taurine (released due to the disruption of peripheral bacterial rhythms) on central clocks.
However, what is relatively clear at present is that there is a mutual regulatory effect between peripheral clocks: clock disruption in one organ will further lead to abnormal expression of clock genes in adjacent organs. We have supplemented relevant content in the original manuscript regarding this mechanism on Page 3, Line 74-77 ” Notably, diet-induced disruption of peripheral clock in colorectal tissue may potentially affect the circadian rhythm stability of other peripheral organs (e.g., liver) via pathways such as cancer cell metastasis, and even feedback to disrupt central clock at the genetic level [17].”

Comments 12: Lines 101–104: Please correct the sentence “…which may resulting in poorer outcome.” to “…which may result in poorer outcomes.”

Response 12: We sincerely appreciate your careful review! We apologize for overlooking this minor detail during the proofreading process and have now made the necessary corrections on Page 4, Line 114-116 ”Upregulation of Wee1 activates Cdc2, leading to phosphorylation of the CDK1/cyclin B complex and subsequent stagnation of cancer cells at the G2/M stage [24], which may result in poorer outcome”.

Comments 13: Lines 161–166: The BMAL1 versus CLOCK influences are compressed into one sentence. I suggest dividing this into two or three sentences and providing more detail on the distinct mechanisms involved.

Response 13: We sincerely appreciate your valuable suggestions! We originally presented Bmal1 and Clock in the same sentence, considering that they function synergistically as a dimer and exhibit differences in primary and secondary functions during the activation of different pathways—but this presentation is indeed prone to misleading readers. In accordance with your suggestions, we have disassembled their functions and elaborated on their mechanism of action more clearly through multiple sentences, enhancing reading fluency on Page 5, Line 183-187 “BMAL1 could stimulate tumor angiogenesis and metastasis, even chemotherapy resistance via binding the E-box of VEGF [9,41,42]. And CLOCK stimulate these pathological processes via interacting with HIF-1α/ARNT to activate the expression of VEGF. Thereby indicate the potential for a novel antimetastatic therapeutic approach.”

Comments 14: Lines 171–175: Please provide a brief description of how BMAL1 influences exosome secretion to strengthen this point; otherwise, the reference to exosomes appears disconnected from the surrounding discussion.

Response 14: Thank you for your valuable suggestions! We have completed the supplementation of content regarding BMAL1 levels regulating the production of specific exosomes to promote CRC metastasis, and made targeted revisions to the preceding and following content, “One study even confirmed that when BMAL1 is highly expressed, the heterodimer formed with CLOCK binds to the E-box elements in the promoter region of the Rab27a gene derived from human CRC cells, thereby transcriptionally activating the expression of Rab27a; this process promotes the secretion of exosomes by CRC cells, and these ex-osomes can further enhance the migration ability of CRC cells and vascular endothelial cells (e.g., HUVECs) in the tumor microenvironment, creating favorable conditions for the invasive metastasis of CRC” on Page 5, Line 192-198, resulting in a more coherent and smooth overall writing logic.

Comments 15: Lines 197–216: This paragraph is overly dense, covering glycolysis, immunity, metastasis, and methodological recommendations. Splitting it into 2–3 thematic paragraphs would greatly improve clarity.

Response 15: Thank you for your valuable suggestions! Our core intention is to emphasize that HKDC1, as a gene that exhibits mutual regulatory interactions with Bmal1, possesses additional functions such as those related to immune evasion, and is expected to serve as a potential therapeutic target in relevant clinical scenarios. Meanwhile, such molecular regulatory characteristics may undergo specific changes in metastatic CRC, and this distinction should be noted in relevant research; bioinformatics studies will further fill this research gap.
Looking back at the original expression, we indeed found that its readability needs improvement. Your suggestion to elaborate by dividing into paragraphs is very pertinent; however, considering that the core focus of this paragraph is metabolic reprogramming, adding new sections focusing on non-core topics such as CRC metastasis may lead to a shift in the focus of the discussion and appear rather abrupt.
Therefore, we have decided to optimize the content and logical order of the original expression, aiming to clearly highlight the aforementioned core points while minimizing impact on overall readability. The specific revised results are as follows: “Recent evidence has shown that Bmal1 plays a negative regulatory role in the glycolytic metabolism of CRC. As mentioned earlier, the activation of the Wnt signaling pathway is mediated by Bmal1 deletion. The additional enhancement of glycolysis is driven by the subsequent upregulation of c-Myc downstream. Both of these effects have been validated in mouse intestinal organoids and CRC patient-derived organoids. The TCGA-COAD database further confirms that CRC patients with this molecular signature (aberrant activation of the Wnt-c-Myc-glycolysis axis) have significantly shorter overall survival (Log-rank P=0.0032) [50]. Based on the aforementioned experimental and clinical evidence, the regulation of the Wnt pathway via clock genes may be the core pathological pathway during the occurrence and development of CRC. 
The hexokinase HKDC1 has been implicated in a variety of gastrointestinal tumors. It is involved in tumorigenesis by regulating glucose and lipid metabolism [51]. Bmal1 and HKDC1 interact and inhibit each other. Perturbed Bmal1 expression induces time-dependent changes in HKDC1 levels and metabolism, with increased glycolytic activity, increased cellular energy supply and proliferation, and a shift toward a meta-static phenotype. The inhibitory effect of Bmal1 on HKDC1 is markedly attenuated in metastatic CRC cells, and the expression and regulatory pathways of clock genes are somewhat altered both in situ and in metastatic CRC cells [10]; thus, in mechanistic studies on clock genes and CRC metabolic reprogramming, it is crucial to focus on the hetero-geneity of gene expression and regulatory mechanisms induced by metastasis. HKDC1 is also involved in various processes, such as immune evasion [52], thus the modulation of the glycolytic pathway involved in HKDC1 by Bmal1 may be essential for patients whose metabolic and metastatic properties are similar to those observed during immune evasion” on Page 6, Line 229-252.

Comments 16: Lines 218–220: This paragraph requires clearer explanation and transitions. Please clarify the mechanistic flow.

Response 16: Thank you for your valuable suggestions. We have reorganized the relevant mechanistic content and potential therapeutic agents suitable for clinical scenarios of colorectal cancer, aiming to provide readers with a clearer reading experience on Page 6, Line 221-228.”CK1δ/ε can enhance the stability and activity of p53 by phosphorylating multiple sites of p53 (e.g., Ser-6, Ser-9, Ser-15, and Ser-20). Subsequently, it directly inhibits aerobic glycolysis by upregulating the downstream target gene TIGAR, reduces glucose uptake by inhibiting the expression of GLUT1, and decreases the shunting of glycolytic inter-mediates to the pentose phosphate pathway by inhibiting the activity of G6PD, ultimately comprehensively inhibiting the aerobic glycolysis process in colorectal cancer cells [49]. Meanwhile, IC261, as a specific inhibitor of CK1δ/ε, is expected to serve as an effective therapeutic agent in relevant clinical scenarios for colorectal cancer [49]“

Comments 17: Lines 327–354: Statements such as “may obtain multiple types of information” are vague. Please specify which markers show the strongest diagnostic or prognostic promise.

Response 17: The question you raised is of great clinical value. After further literature retrieval, we regretfully found that there is no specialized systematic summary of clock genes as prognostic biomarkers for CRC in existing literature—this has only been verified in TCGA database analyses and other related studies, where multiple clock genes are closely associated with the diagnosis and prognosis of various cancers including CRC, with relatively consistent research results: namely, the expression levels of most clock genes show a positive correlation with cancer prognosis, while the expression level of the TIM gene exhibits a negative correlation with prognosis. Based on the above findings, we have revised the relevant literature descriptions. Meanwhile, we have optimized the logical flow of this paragraph, making the text more concise and accurate. “Multiple analyses based on The Cancer Genome Atlas (TCGA) database have confirmed that various clock genes are closely associated with the prognosis of cancer patients. Although there are currently no specialized prognostic prediction analyses for clock genes in CRC, multiple studies on non-small cell lung cancer and other cancers have revealed certain commonalities, core clock genes such as BMAL1 and PER2 show a positive cor-relation between their expression levels and patient prognosis, while the expression level of the TIM gene exhibits a negative correlation with prognosis. As diagnostic and prognostic prediction indicators, clock genes exhibit good accuracy, with the area under the curve (AUC) reaching 0.8 in some analytical models, which is superior to traditional detection indicators (e.g., CEA, CA199, and CA125) [76,77]. The expression of clock genes in CRC exhibits location-related heterogeneity. Existing study has shown that the ex-pression difference of Cry1 has more significant prognostic predictive value in female patients. Specifically, in female patients with right-sided colon CRC, the expression level of Cry1 in tumor tissues is significantly higher than that in adjacent tissues, and this high Cry1 expression is negatively correlated with patient prognosis; in contrast, no significant expression difference of such clock genes (i.e., Cry1, Cry2) is observed between tumor tissues and adjacent tissues in female patients with left-sided CRC [78]. In summary, by detecting clock gene expression levels, we may obtain multiple types of information, such as the TNM stage of CRC, degree of malignancy and the prognosis to a certain extent. Although there is still a problem of an insufficient sample size for testing, further expansion of the number of clinical tests will better extract useful information from the clock gene level and provide more accurate and simple diagnostic methods” on Page 9, Line 380-402.

Comments 18: Conclusions: Several limitations repeat earlier statements. This section would benefit from being condensed and structured around: (1) established mechanisms; (2) emerging connections; (3) clear research priorities; and (4) translational challenges and opportunities. This would make the conclusion more impactful.

Response 18: We sincerely appreciate your valuable suggestions! It is well-founded that you pointed out the issue of repetition and redundancy in the Conclusion section. In accordance with your suggestions, we have conducted systematic revisions from four dimensions: (1) established mechanisms, (2) emerging connections, (3) clear research priorities, and (4) translational challenges and opportunities.
In the revision process, while fully retaining the core fundamental information, we added several innovative perspectives to strengthen the logical connections between various sections and deepen the discussion on the core value of research on clock genes and CRC, making the Conclusion section more in-depth and speculative than the previous version. The specific revised content is as follows on Page 12, Line 488-547: ”In summary, the role of clock genes in CRC has been clarified to a certain extent. Clock genes expressed in a normal rhythm are crucial for maintaining the homeostasis of cellular physiological activities. When the expression rhythm of clock genes is disrupted, it directly triggers a series of pathological alterations, such as abnormal regulation of the cell cycle, imbalance of cellular metabolism, and formation of the tumor microenvironment as summarized in Table 1. These pathological characteristics mediated by clock gene dysregulation have been confirmed to serve as potential biomarkers for CRC diagnosis and core targets for therapeutic intervention [6,76,77,91], providing a clear molecular basis for the precision diagnosis and treatment of CRC.
Furthermore, in the field of traditional therapy, research related to clock genes has also spawned new therapeutic ideas. Combining classic conventional CRC treatment regi-mens (e.g., chemotherapy, immunotherapy) with chronotherapy—regulating drug administration time based on the rhythm of clock genes—can significantly improve therapeutic efficacy and reduce toxic side effects [65,81-85]. This interdisciplinary combination offers a new direction for breaking through the bottlenecks of traditional therapy and has demonstrated promising potential in some preliminary studies as illustrated in Table 2. 
Notably, current research on clock genes in CRC is still in its initial stage, and future efforts need to focus on two core directions. The primary focus is to thoroughly dissect the molecular mechanisms by which clock genes regulate the occurrence and development of CRC, identify key functional nodes in the pathological process, and on this basis, develop technological approaches such as synthetic biology to achieve direct targeted therapy against clock gene-related targets. The secondary focus is to clarify the impact of the circadian clock on the mechanisms of traditional drug therapy, and optimize existing administration protocols and treatment processes through rhythm regulation to further enhance the precision and effectiveness of CRC treatment. 
Additionally, there are two other research directions that have been relatively less explored but still hold great translational potential. Based on the potential synergy between gut microbiota-based interventions and chronotherapy mentioned earlier, this combined approach can be more easily translated into clinical practice. Time-restricted feeding (TRF) can reshape colonic circadian rhythms by regulating gut microbiota composition (e.g., restoring SCFA-producing bacteria) [6]. Supplementing SCFA-producing probiotics (e.g., Bifidobacterium breve) or prebiotics (e.g., inulin) can restore the circadian rhythms of microbial metabolites and intestinal clocks. When combined with chronotherapy, ad-ministering REV-ERBα agonists during peak inflammatory periods can synergistically inhibit NLRP3-mediated inflammation and further enhance anti-tumor immunity [68]. Existing studies have shown that epigenetic modifications targeting clock genes (such as methylation and histone acetylation) can also exert regulatory effects on various down-stream physiological processes, including metabolism and cell cycle. Therefore, subsequent studies on the epigenetic modifications of clock genes will also be one of the highly promising research avenues [101,102].
To seize these opportunities, three core bottlenecks must be overcome. At the animal model level, mammalian models should be prioritized, and the differences in the active periods of the biological clock between model animals and humans must be strictly corrected to ensure the clinical applicability of research results. At the detection and clinical management level, therapeutic efficacy evaluation indicators need to be refined to a more precise temporal dimension; meanwhile, in clinical practice, patients’ adherence to rhythmic treatment regimens should be strengthened to avoid efficacy impairment due to temporal deviations. At the research design level, factors influencing circadian clock heterogeneity (such as gender and age) should be included in clinical cohort analyses to clarify their regulatory effects on the heterogeneity of treatment outcomes, thereby providing a basis for formulating individualized treatment plans.
Although clock genes face numerous challenges in clinical translation for CRC, their great potential in diagnosis, treatment, and regimen optimization has been clearly demonstrated. If the aforementioned bottlenecks can be broken through, it will surely bring a revolutionary leap to the field of CRC diagnosis and treatment.”

Comments 19: Figure 1: The caption should more clearly explain the interactions between environmental cues and the TTFL system.

Response 19: We appreciate your detailed and supplementary comments on Figure 1. Based on these suggestions, we have refined the figure legend “Environmental cues regulate the transcription-translation feedback loop of the circadian clock via direct and indirect mechanisms. Light directly stimulates the SCN, altering the central circadian clock. In contrast, food modulates intestinal flora and gastrointestinal cells, whose secretions thereby regulate the colorectal peripheral clock and influencing the synchronization between the central and peripheral clocks” on Page 2, Line 57-61 with the aim of providing readers with a clearer reading experience.

Comments 20: Figure 2: Although an effective visual summary, please consider referencing it explicitly in the clinical sections for greater integration.

Response 20: Thank you for your valuable suggestions. We initially placed Figure 2 in the conclusion section, intending to guide readers to review the core mechanisms of the entire article again at the end. However, this arrangement may indeed overlook the coherence and readability between paragraphs. Following your suggestions, we have moved forward the first presentation of Figure 2 to the introductory part of the clinical Section, on Page 9, Line 360, with the aim of helping readers clearly understand the specific focus of the clinical applications of this study.

Comments 21: Tables 1 and 2: Some fields use “unmentioned” and “not mentioned” inconsistently. Please standardize terminology.

Response 21: We appreciate your correction. We have identified the inconsistent use of "not mentioned" and "unmentioned" on Page 13, Table 2, and have replaced "not mentioned" with "unmentioned" to ensure consistency with the previous context. We would like to thank you again for your time and meticulous attention to detail.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript entitled “Circadian Clock Genes in Colorectal Cancer: From Molecular  Mechanisms to Chronotherapeutic Applications” highlighted associations between circadian disruption and CRC, including diagnostic markers, prognostic assessment, and chemosensitivity.

 Colorectal cancer (CRC) is associated with genetic mutation and other carcinogenic and metabolomic factors, but does not directly involve Circadian Clock Genes. Circadian Clock Genes may influence the initiation and progression of cancer. The article has been written in a very diffuse and superficial manner, lacking the fundamental compressive mechanism and outcomes.

Comments on the Quality of English Language

The manuscript entitled “Circadian Clock Genes in Colorectal Cancer: From Molecular  Mechanisms to Chronotherapeutic Applications” highlighted associations between circadian disruption and CRC, including diagnostic markers, prognostic assessment, and chemosensitivity.

 Colorectal cancer (CRC) is associated with genetic mutation and other carcinogenic and metabolomic factors, but does not directly involve Circadian Clock Genes. Circadian Clock Genes may influence the initiation and progression of cancer. The article has been written in a very diffuse and superficial manner, lacking the fundamental compressive mechanism and outcomes.

Author Response

Comments 1: The manuscript entitled “Circadian Clock Genes in Colorectal Cancer: From Molecular  Mechanisms to Chronotherapeutic Applications” highlighted associations between circadian disruption and CRC, including diagnostic markers, prognostic assessment, and chemosensitivity.

Response 1: Thank you for your valuable summary! We have further supplemented content related to potential translational entry points for the future, aiming to further enhance the logical coherence and academic value of the manuscript.

Comments 2: Colorectal cancer (CRC) is associated with genetic mutation and other carcinogenic and metabolomic factors, but does not directly involve Circadian Clock Genes. Circadian Clock Genes may influence the initiation and progression of cancer. The article has been written in a very diffuse and superficial manner, lacking the fundamental compressive mechanism and outcomes.

Response 2: Your comments hold merit. There is currently no direct evidence that clock genes can directly induce the development of CRC. Most existing relevant studies have involved additional interventions on clock genes in established CRC animal models, and such manipulations often lead to more malignant phenotypes. Therefore, we have explicitly revised the statement in the manuscript to "clock genes typically participate in the initiation and progression of CRC" to align with the objective evidence from current research.
Meanwhile, we have conducted a more in-depth analysis of the existing issues and potential value in multiple studies, and supplemented research progress in epigenetic regulation and other related fields, aiming to reverse your impression of this manuscript's lack of depth. Regarding the issue you pointed out that the mechanism descriptions are relatively scattered, we have conducted systematic revisions and integration of multiple relevant sections in the original text, aiming to significantly enhance the logical coherence of the review and provide references and insights for research in this field. Additionally, in the Conclusion section, we not only summarized the core mechanisms but also proposed several of the most research-worthy translational directions, and we hope this meets your expectations.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have provided an excellent and thorough response to the previous major revision requirements, transforming a manuscript with substantial inconsistencies into a coherent, well-structured, and scientifically robust review. Key improvements include the harmonization of genetic nomenclature, clarification of mechanistic contradictions, and expansion of mechanistic depth, particularly in the sections on tumour microenvironment and chronotherapy. Overall, the manuscript is significantly strengthened and is now close to publication.

I recommend acceptance after minor revisions, aimed at polishing clarity, consistency, and translational relevance without altering the core scientific content.

Remaining points to address:

• References: please verify the currency and accuracy of newly added citations and provide DOIs where missing. If available, consider integrating recent (2022–2025) meta-analyses or cohort studies addressing circadian genes in colorectal cancer.
• Figures and Tables: in Figure 1, please spell out “SCN (Suprachiasmatic Nucleus)” for clarity. In Table 1, please standardize the use of “N/A” instead of “Unmentioned.” Ensure all figures meet journal requirements (>300 dpi). Figure 2 should be explicitly referenced in the main text (Sections 3.1 or 3.2) to better integrate its conceptual summary with the manuscript narrative.
• Nomenclature: a few instances of “Bmal1” remain. Please standardize all human gene names to uppercase (e.g., BMAL1) throughout.
• Mechanistic clarifications: please add 1–2 sentences clarifying the mechanistic hierarchy among core clock genes (e.g., BMAL1/CLOCK as upstream regulators relative to PER/CRY/TIM feedback components), as this will greatly improve conceptual synthesis for readers.
• Chronotherapy and chemoresistance: this section would benefit from a brief expansion (2–3 sentences) addressing circadian regulation of DNA repair pathways (e.g., NER, PARP1 rhythms) and drug metabolism (e.g., CYP oscillations), which constitute the mechanistic basis for chronotherapeutic optimization.
• CRC heterogeneity: the discussion of CRY1 heterogeneity is appreciated. However, adding a short paragraph on how CRC molecular subtypes (e.g., CMS1–CMS4) may modulate circadian gene expression or chronotherapy responsiveness would enhance translational relevance.
• Paragraphs remaining overly dense: some sections (particularly the HKDC1-related paragraph and the REV-ERB/NLRP3 discussion) remain overly dense and would benefit from further simplification and clearer transitions.
• Conclusion: although improved, the conclusion remains lengthy. Condensing it into four focused themes/paragraphs (established mechanisms; emerging connections; research priorities, and translational challenges/opportunities) would enhance clarity and impact.

Author Response

Comments 1: References: please verify the currency and accuracy of newly added citations and provide DOIs where missing. If available, consider integrating recent (2022–2025) meta-analyses or cohort studies addressing circadian genes in colorectal cancer.

Response 1: We appreciate your kind reminder! We have verified that the DOI number for Reference 12 was missing and have now supplemented it in full. “[12]    Huang, C., Zhang, C., Cao, Y., Li, J., & Bi, F., Major Roles of the Circadian Clock in Cancer, 2023, Cancer Biol. Med., 20, 1, 10.20892/j.issn.2095-3941.2022.0474”. Regarding the meta-analysis focusing on the role of clock genes in CRC during 2022–2025 mentioned before, we have not found any relevant reports to date. The latest meta-analysis investigating the association between clock genes and cancer was published in 2017, which only briefly covered CRC (with only one relevant case study). Although no specific mechanism of clock gene regulation in CRC or high-quality evidence for associations has been identified, this study confirmed a potential association between clock gene dysregulation and CRC risk.

Comments 2: Figures and Tables: in Figure 1, please spell out “SCN (Suprachiasmatic Nucleus)” for clarity. In Table 1, please standardize the use of “N/A” instead of “Unmentioned.” Ensure all figures meet journal requirements (>300 dpi). Figure 2 should be explicitly referenced in the main text (Sections 3.1 or 3.2) to better integrate its conceptual summary with the manuscript narrative.

Response 2: We sincerely appreciate your kind reminder! We noticed that the full name of SCN was omitted in the legend of Figure 1 and have now supplemented it completely. Subsequently, in accordance with your requirements ”Light directly stimulates the Suprachiasmatic Nucleus (SCN), altering the central circadian clock”, on Page 2, Line 59. We have replaced all instances of “Unmentioned” in the tables with “N/A”. Meanwhile, we have confirmed that the resolution of both figures meets the journal’s requirements. In addition, when clock genes are first mentioned as diagnostic and prognostic biomarkers in Section 3.1, we have cited Figure 2 “Multiple analyses based on TCGA database have confirmed that various clock genes are closely associated with the prognosis of cancer patients like in Figure 2”on Page 10 Line 375-376; when elaborating on the definition of chronotherapy in Section 3.2, we have referenced the same figure again “All these factors lead to chronotherapy, a therapeutic approach that adjusts the timing of chemotherapeutic drug administration according to biological rhythms, primarily the circadian clock, as illustrated in Figure 2”, on Page 11, Line 420-422, aiming to enhance the logical flow and clarity of the manuscript.

Comments 3: Nomenclature: a few instances of “Bmal1” remain. Please standardize all human gene names to uppercase (e.g., BMAL1) throughout.

Response 3: We sincerely appreciate your careful correction. We have uniformly revised “Per2” to “PER2” in the original manuscript on Page 8, Line 336. Regarding the capitalization of “Bmal1”, after rechecking, we did not find any incorrect lowercase usage in the text. As “Bmal1” appears as the original expression in the title of the cited reference, we have retained its original form without modification and would like to inform you of this situation.

Comments 4: Mechanistic clarifications: please add 1–2 sentences clarifying the mechanistic hierarchy among core clock genes (e.g., BMAL1/CLOCK as upstream regulators relative to PER/CRY/TIM feedback components), as this will greatly improve conceptual synthesis for readers.

Response 4: We sincerely appreciate your valuable suggestions! When BMAL1/CLOCK were first introduced in the original manuscript, their status as core regulators has been clearly specified “Basic helix–loop–helix ARNT like 1 (BMAL1) and its partner, circadian locomotor output cycle protein kaput (CLOCK), are the main regulators of the loop; they work as a tran-scription factor that drive the positive arm of the loop by heterodimerization” on Page 2 Line 42-44. However, this description may appears relatively early, at which point readers have not yet formed a clear overall understanding of the TTFL, which may lead to misunderstandings. Therefore, after fully introducing the core components of TTFL, we have added a brief summary of the relevant content mentioned earlier, aiming to deepen readers’ impressions and facilitate their comprehensive understanding of the TTFL regulatory network on Page 2 Line 51-54 “In summary, as core positive regulators of the TTFL pathway, BMAL1/CLOCK can regulate downstream PER/CRY/TIM feedback components, thereby modulating other downstream genes and related physiological processes. Thus, disorders of circadian rhythm may affect a wide range of ailments, e.g., caners, especially CRC [6-8]”.

Comments 5: Chronotherapy and chemoresistance: this section would benefit from a brief expansion (2–3 sentences) addressing circadian regulation of DNA repair pathways (e.g., NER, PARP1 rhythms) and drug metabolism (e.g., CYP oscillations), which constitute the mechanistic basis for chronotherapeutic optimization.

Response 5: Thank you for your suggestions. The drugs mentioned in our manuscript, such as 5-fluorouracil (5-FU), exert their cytotoxic effects primarily by replacing normal pyrimidines and disrupting the normal physiological function of DNA. They do not involve DNA repair processes nor are they related to the CYP enzyme family you mentioned; furthermore, after searching relevant references, we did not find relevant evidence to support these contents, so we did not supplement them. However, we understand that the core of your suggestion is to provide specific mechanistic explanations or application examples for the theory of chronotherapy. Therefore, we have expanded the content related to 5-FU-based chronotherapy, detailed supplemented the selection of its administration time, the specific theories and relevant scientific data on which it is based on Page 11 Line 423-431 “The proportion of cells in the DNA synthesis phase exhibits a 24-hour circadian rhythm, with peak levels observed between 8:00 and 20:00 in various tissues such as bone marrow and intestines. Meanwhile, the activity of the rate-limiting enzyme for fluorouracil me-tabolism, dihydropyrimidine dehydrogenase (DPD), peaks between 22:00 and 00:00. This circadian characteristic enables daytime administration to maximize the inhibitory effect on cancer cell proliferation [82]. Therefore, aligning drug administration with the 24-hour rhythms of host drug metabolism-related enzyme activities and target cell killing mechanisms not only enhances therapeutic efficacy but also reduces tumor drug resistance while minimizing toxic side effects on normal tissues”, Page 12 Line 478-483 “In 5-FU chemotherapy, BMAL1 levels peak during the dark phase of mouse, and the activity of 5-FU metabolic enzymes regulated by BMAL1 (such as UMPS, UCK2, and UPP2) also exhibits a highly synchronized circadian rhythm. Administering 5-FU during this time period significantly enhances its therapeutic efficacy [74]. Similarly, the ex-pression rhythm of PER1 shows high synchronization with that of DPD during the dark phase” aiming to deepen readers’ understanding of chronotherapy.

Comments 6: CRC heterogeneity: the discussion of CRY1 heterogeneity is appreciated. However, adding a short paragraph on how CRC molecular subtypes (e.g., CMS1–CMS4) may modulate circadian gene expression or chronotherapy responsiveness would enhance translational relevance.

Response 6: We sincerely appreciate your suggestions. We recall that in your first-round comments, you proposed clarifying the CRC molecular subtypes involved in each section and their characteristics. However, the vast majority of existing literature has not elaborated on the molecular subtypes of the models used. This time, we found a brief discussion on differences in clock gene expression among various CMS subtypes in a 2023 study focusing on circadian rhythm timeline algorithm analysis, which has been supplemented after the chronotherapy-related section. This is aimed at making readers aware that not all types of CRC are suitable for administration at a single fixed time window on Page 12 Line 490-496, ”Notably, there are significant differences in the rhythmic expression of genes among the four consensus molecular subtypes (CMS1-4): the peak of circadian oscillation in CMS1 is concentrated at the 9-hour phase with higher TIM gene expression; in contrast, the peak of circadian oscillation in CMS4 is enriched at the 3-hour phase, characterized by low TIM expression and high ZEB1 expression [93]. This difference not only explains the high metastatic potential of CMS4 observed clinically but also indicates that chronotherapeutic strategies should vary among different subtypes.” 

Comments 7: Paragraphs remaining overly dense: some sections (particularly the HKDC1-related paragraph and the REV-ERB/NLRP3 discussion) remain overly dense and would benefit from further simplification and clearer transitions.

Response 7: We agree with your valuable suggestions. As this part of the research is relatively in-depth and covers a wide range of content, the discussion section formed after integration is inevitably somewhat lengthy. However, we are reluctant to omit too much valid information; therefore, after streamlining redundant and complex expressions, we have focused on optimizing the presentation logic of the content on Page 6 Line 222-251 “Specifically, CK1δ/ε enhances the stability and activity of p53 by phosphorylating multiple sites (e.g., Ser-6, Ser-9, Ser-15, and Ser-20). It then directly inhibits aerobic glycolysis by upregulating the downstream target gene TIGAR, reduces glucose uptake by sup-pressing GLUT1 expression, and decreases the shunting of glycolytic intermediates to the pentose phosphate pathway by inhibiting G6PD activity, ultimately comprehensively suppressing the aerobic glycolysis process in CRC cells [49]. As a specific inhibitor of CK1δ/ε, IC261 is expected to serve as an effective therapeutic agent in relevant clinical scenarios for CRC [49].
Moreover, the core clock gene BMAL1 plays a negative regulatory role in the glycolytic metabolism of CRC. As mentioned earlier, BMAL1 deletion mediates the activation of the Wnt signaling pathway, which further upregulates downstream c-Myc to drive enhanced glycolysis—this effect has been validated in mouse intestinal organoids and CRC patient-derived organoids. The TCGA-COAD database further confirms that CRC patients with this molecular signature (aberrant activation of the Wnt-c-Myc-glycolysis axis) have significantly shorter overall survival (Log-rank P=0.0032) [50], suggesting that clock gene-mediated regulation of the Wnt pathway may be a core pathological pathway in the initiation and progression of CRC. 
Hexokinase HKDC1 is implicated in various gastrointestinal tumors and participates in tumorigenesis by regulating glucose metabolism [51]. BMAL1 and HKDC1 exert mutual inhibition: perturbed BMAL1 expression induces time-dependent changes in HKDC1 levels and metabolism, characterized by increased glycolytic activity, enhanced cellular energy supply, and a shift toward a metastatic phenotype. In metastatic CRC cells, the inhibitory effect of BMAL1 on HKDC1 is markedly attenuated, and the expression of clock genes and their regulatory pathways differ somewhat between primary and metastatic CRC cells [10]. Thus, in mechanistic studies on clock genes and CRC metabolic reprogramming, it is crucial to focus on the heterogeneity of gene expression and regulatory mechanisms induced by metastasis. Notably, HKDC1 is also involved in processes such as immune evasion [52]; therefore, BMAL1-mediated modulation of the HKDC1-related glycolytic pathway may be essential for CRC patients whose metabolic and metastatic properties are associated with immune evasion”, Page 8 Line 298-307 “In the DSS-induced mouse colitis model, REV-ERBα inhibits the NLRP3 inflammasome via a dual mechanism: first, it specifically binds directly to the inflammasome’s promoter region to suppress transcription; second, it inhibits p65 and its downstream NF-κB pathway, indirectly repressing NLRP3 and attenuating macrophage-mediated inflammatory responses. The anti-inflammatory effect of the REV-ERBα agonist SR9009 in this pathway has been validated in animal models [68]. Based on this mechanism, a nanolipid carrier (NLC) hydrogel enriched with high galacturonic acid pectin (i.e., modified citrus pectin, MCP4) and loaded with 6-gingerol (6G) has been developed. It targets inflammatory sites and downregulates NLRP3 activation by regulating the NF-κB inflammatory pathway and REV-ERBα/β [69]”, aiming to provide readers with a smoother reading experience. 

Comments 8: Conclusion: although improved, the conclusion remains lengthy. Condensing it into four focused themes/paragraphs (established mechanisms; emerging connections; research priorities, and translational challenges/opportunities) would enhance clarity and impact.

Response 8: We sincerely appreciate your valuable suggestions! Previously, the conclusion section had issues such as redundant expressions and overly detailed descriptions, making it somewhat lengthy. Therefore, we have streamlined the content related to the confirmed mechanisms of clock genes and merged the original two paragraphs into one. Subsequently, we adjusted the presentation order of research opportunities, core future research directions, and other content to align with your suggestions, on Page 12 Line 498-534 “Clock genes’ role in CRC has been partially clarified. Disrupted expression rhythms of clock genes directly trigger a series of pathological alterations as illustrated in Table 1, making them potential biomarkers for CRC therapeutic intervention [6,76,77,93]. Additionally, combining classic CRC treatments (e.g., chemotherapy, immunotherapy) with chronotherapy significantly improves efficacy and reduces toxic side effects, with promising preliminary evidence as illustrated in Table 2 [65,81-85].
Notably, two under-explored but translationally valuable connections deserve attention: first, the synergistic effect of gut microbiota-based interventions and chronotherapy facilitates clinical translation, as time-restricted feeding (TRF) reshapes colonic circadian rhythms by regulating microbiota composition (e.g., restoring SCFA-producing bacteria) [6], while supplementing SCFA-producing probiotics/prebiotics (e.g., Bifidobacterium breve, inulin) restores microbial metabolite and intestinal clock rhythms; second, epi-genetic modifications (e.g., methylation, histone acetylation) targeting clock genes regulate downstream processes (e.g., metabolism, cell cycle), representing a promising re-search direction [101,102]. 
Future research should focus on two core directions: first, identify key functional nodes in the pathological process by which clock genes regulate CRC initiation and progression, and develop synthetic biology-based technologies to achieve direct targeted therapy against clock gene-related targets; second, clarify the circadian clock’s impact on traditional drug therapy mechanisms, and optimize administration protocols and treatment processes via rhythm regulation to enhance the precision and effectiveness of CRC treatment.
Clock genes have demonstrated great potential in CRC diagnosis, treatment, and regimen optimization. However, three core bottlenecks must be addressed. At the animal models level, prioritize mammalian models and correct circadian active period differences between animals and humans to ensure clinical applicability; at the detection and clinical management level, refine efficacy indicators to a precise temporal dimension and strengthen patient adherence to rhythmic regimens to avoid efficacy impairment from temporal deviations; at the research design level, incorporate circadian heterogeneity-related factors (e.g., gender, age) into clinical cohort analyses to clarify their regulatory effects on treatment outcomes, laying a foundation for personalized plans.
Breaking through these bottlenecks will bring a revolutionary leap to CRC diagnosis and treatment.” We hope this meets your expectations.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The revised manuscript entitled “Circadian Clock Genes in Colorectal Cancer: From Molecular Mechanisms to Chronotherapeutic Applications” is quite interesting and comprehensively revised. The authors have improved the manuscript well. The manuscript can be accepted for publication.   

Author Response

Comments 1: The revised manuscript entitled “Circadian Clock Genes in Colorectal Cancer: From Molecular Mechanisms to Chronotherapeutic Applications” is quite interesting and comprehensively revised. The authors have improved the manuscript well. The manuscript can be accepted for publication.

Response 1: We sincerely appreciate your affirmation! Following your valuable suggestions as well as those from other reviewers, we have further reviewed and optimized the references, image resolution, and other aspects of this manuscript. Meanwhile, we have supplemented content including the regulatory mechanisms of BMAL1 and CLOCK (core clock genes) on downstream genes, as well as the heterogeneity of clock genes in colorectal cancer (CRC) of different CMS subtypes. We aim to meet the journal's submission requirements while providing higher academic value to readers.

 

Author Response File: Author Response.docx