The Relevance of Circadian Clocks to Stem Cell Differentiation and Cancer Progression

Round 1

Reviewer 1 Report

The manuscript by Malik et al. provides basic knowledge in circadian clocks, stem cells and cancer progression. Circadian phenomena in different stages of stem cells (differentiation and maturation) and cancer cells (proliferation, metastasis and invasion) are comprehensively summarized and discussed. In addition, the authors point out the limitations of the existing researches and present their perspective. I think the manuscript is well structured and written and can be accepted for publication.

The panels A-I should be marked in Figure 2.

Author Response

Response: We thank the reviewer for their constructive comments and the time to review our publication. We have updated our Figure 2 in our current resubmission and fixed the error.

Reviewer 2 Report

Summary:

The authors of this research article are investigating the relevance of circadian clock in stem cell differentiation and cancer progression, focusing on the differentiation of adult neural stem cells and cancer stem cells to determine how cells on several differentiation stages could harbor unique states of molecular circadian clocks that can lead to different outcomes in a cell differentiation and its eventual fates. The authors argued that, while disorganized circadian function or loss can increase cancer risk, it could not be the only contributor, considering how the figure the authors presented did not indicate any form of circadian clock regulation during cell differentiation. However, while the paper focused on circadian clock regulation, the authors should make clear on what kind of stimuli or external factors that can affect cell differentiation and circadian regulation, and why in-vitro studies are preferable and how it can be eventually applied to in-vivo studies.

Major comments.

1. The authors made a good argument on the relevancy of many of the studies the paper reviewed and managed to explain why there is a lack of circadian clock function in figure 2. However, the figure itself lacks any indication of its relationship to circadian function. At a glance, the figure only explains the forming of neural stem cells and how it can differentiate into cancer stem cells and metastasize throughout the body. It has nothing to do with circadian functions despite the description.
2. The description for figure 2 specified that it is visually describing the Circadian properties of stem cells and their lineages during development and cancer, but then further description of the figure, specifically lines 166 to 192, has nothing to do with the figure itself. There is no way to determine if anything described between lines 166 and 192 is related to Figure 2.
3. Page 14: In 5. Conclusion, the authors specifically considered that in vitro studies might give a satisfying result as the rhythms were easily tracked and not affected by rhythms of multiple origins and to possibly determine the exact cause of cancer progression through observation of circadian rhythms. However, previously, they have also argued that there is no one specific way to determine why cancers can form, while also arguing that circadian disruption is not a sufficient causal factor.
4. Many of the points emphasizing the relevancy of circadian functions and its loss to the development of cancer stem cells have been described as experimental and based on assumptions and possibilities. The author should consider finding more hard evidences to corroborate the relevancy of circadian functions in the relationship between neural stem cells and cancer stem cells.

Minor comments.

1. For paragraphs including Page 4, Line 150, Page 12, Line 526: a recent article (PMID: 34930826) can be cited because this shows a piece of evidence for the core clock function in embryonic development.
2. Page 2, Line 96: REV-ERB should be REV-ERB.
3. Page 3, Line 107: ROE should be RORE.
4. Page 4, Line 115: Please correct “..this clock output. Circadian rhythms [9]. Rhythms…”
5. Page 9, Line 366: Please correct  “…from TTFLs [53].consisting of an array…”
6. Page 9, Line 540-542; it sounds awkward, “Assays for circadian rhythms in samples from whole tumors taken from multiple animals over time or continuous measurements of bioluminescence signals from tumors or cell cultures would have been compromised for the same reasons.”
7. Page 11-12, Lines 493-502; Uneven font sizes
8. Line 505: Possible double spacing between full stop and CTC
9. Line 520: Double Spacing between ‘cells’ and ‘resembling’
10. Line 648-649: It would be better if the start of the Reference is moved down one page. The first reference can easily be missed.

Author Response

Reviewer 2:

The authors of this research article are investigating the relevance of circadian clock in stem cell differentiation and cancer progression, focusing on the differentiation of adult neural stem cells and cancer stem cells to determine how cells on several differentiation stages could harbor unique states of molecular circadian clocks that can lead to different outcomes in a cell differentiation and its eventual fates. The authors argued that, while disorganized circadian function or loss can increase cancer risk, it could not be the only contributor, considering how the figure the authors presented did not indicate any form of circadian clock regulation during cell differentiation. However, while the paper focused on circadian clock regulation, the authors should make clear on what kind of stimuli or external factors that can affect cell differentiation and circadian regulation, and why in-vitro studies are preferable and how it can be eventually applied to in-vivo studies.

Response: We thank the reviewer for their constructive comments and the time to review our publication. We have updated our current resubmission and indicated how circadian regulation occurs at certain stages, and we fixed the error.

Major comments.

1. The authors made a good argument on the relevancy of many of the studies the paper reviewed and managed to explain why there is a lack of circadian clock function in figure 2. However, the figure itself lacks any indication of its relationship to circadian function. At a glance, the figure only explains the forming of neural stem cells and how it can differentiate into cancer stem cells and metastasize throughout the body. It has nothing to do with circadian functions despite the description.  

Response: We thank the reviewer for their constructive comments. We have updated our Figure 2 and indicated how circadian regulation appears to be present at different stages of differentiation, etc.

1. The description for figure 2 specified that it is visually describing the Circadian properties of stem cells and their lineages during development and cancer, but then further description of the figure, specifically lines 166 to 192, has nothing to do with the figure itself. There is no way to determine if anything described between lines 166 and 192 is related to Figure 2.

Response Thank you for the comment. We have updated our Figure 2 and indicated where circadian regulation is likely to be present at different stages according to the evidence that is discussed further in the text. Corrections were made in the figure 2 legend.

1. Page 14: In 5. Conclusion, the authors specifically considered that in vitro studies might give a satisfying result as the rhythms were easily tracked and not affected by rhythms of multiple origins and to possibly determine the exact cause of cancer progression through observation of circadian rhythms. However, previously, they have also argued that there is no one specific way to determine why cancers can form, while also arguing that circadian disruption is not a sufficient causal factor.

Response: We added clarification that cancer progression depends on multiple factors and that the contributions from circadian rhythms can be evaluated using in vitro methods, at lines 597-604 and 650-654. Because distinct circadian rhythms proceed in some cancer cells, including gliomas, it is not possible to say that loss or near loss of rhythmicity is necessary for cancer progression in all cases. It is possible that circadian disruption is a causal factor in combination with other factors known to contribute to cancer cell proliferation and progression to the next stage. To address this complexity the interactions between factors would be most easily tested in vitro where the relative importance of a strong or weak circadian rhythm can be measured. It seems unlikely that circadian disruption alone is a sufficient factor on its own, as we stated on line 602, because cancer incidences probably would be much higher when considering the pervasiveness of chronic sleep disruption and other circadian disturbances in the human population. For example, glioblastoma remains a rare cancer in our modern society. We are using “sufficient” here as when describing a neural circuit that when activated is sufficient to produce a particular behavior, even when other circuits might also be sufficient to produce the behavior.

Many of the points emphasizing the relevancy of circadian functions and its loss to the development of cancer stem cells have been described as experimental and based on assumptions and possibilities. The author should consider finding more hard evidences to corroborate the relevancy of circadian functions in the relationship between neural stem cells and cancer stem cells.

Response: We included additional evidence of the relationship between circadian rhythms and stem cells at lines 443-449 and 514-516. We cite the possible origins of CSCs in NSCs at lines 708-709, but there appears to be no hard evidence available on how the circadian timing system may control this process.

Minor comments.

1. For paragraphs including Page 4, Line 150, Page 12, Line 526: a recent article (PMID: 34930826) can be cited because this shows a piece of evidence for the core clock function in embryonic development.

Proc Natl Acad Sci U S A. 2022 Jan 4;119(1):e2114083119. Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids Yasuhiro Umemura  1 , Nobuya Koike  1 , Yoshiki Tsuchiya  1 , Hitomi Watanabe  2 , Gen Kondoh  2 , Ryoichiro Kageyama  3 , Kazuhiro Yagita  4

              Response: Thanks. Added it.

1. Page 2, Line 96: REV-ERB should be REV-ERB. Corrected
2. Page 3, Line 107: ROE should be RORE. Corrected
3. Page 4, Line 115: Please correct “..this clock output. Circadian rhythms [9]. Rhythms…” Fixed. Thanks
4. Page 9, Line 366: Please correct  “…from TTFLs [53].consisting of an array…”  Corrected
5. Page 9, Line 540-542; it sounds awkward, “Assays for circadian rhythms in samples from whole tumors taken from multiple animals over time or continuous measurements of bioluminescence signals from tumors or cell cultures would have been compromised for the same reasons.”

We improved this sentence.

1. Page 11-12, Lines 493-502; Uneven font sizes. Corrected
2. Line 505: Possible double spacing between full stop and CTC. Corrected
3. Line 520: Double Spacing between ‘cells’ and ‘resembling’. Corrected
4. Line 648-649: It would be better if the start of the Reference is moved down one page. The first reference can easily be missed. Corrected

Reviewer 3 Report

The review by Malik et al focusses on the role of the molecular clockwork on adult neurogenesis and glioma cancer stem cells. In addition, the role of the circadian system and the relevance of circadian misalignment on tumor development and progression as well as cancer chronotherapy are briefly discussed. The topic is very interesting and highly relevant. However, the different levels of regulation are not very clearly defined. The article would benefit greatly if the authors paid more attention to the role of systemic rhythmic factors for regular and/or rhythmic cell function.

Major

• Please indicate in the title/abstract and/or the first part of the introduction that you focus on vertebrates/mammals
• The definition of the terms “circadian clock” or “clock” is not quite clear. On page 2, the circadian clock is defined as multicellular circadian oscillators, eg in SCN or peripheral oscillator. In Figure 1, the circadian clock seems to be equal to the molecular circadian clockwork. On page 10 ( lane 418) the circadian clock seems to be equal to the circadian system: “melatonin is under circadian clock control”, similarly, page 13 lane 569: “VEGF signaling cascades and components that regulate angiogenesis are 569 under clock control”. Please clearly define the components and their effects at the molecular/cellular, multicellular and systemic level.
• Please explain how rhythmic systemic factors such as metabolic signals, light at night and chronic stress, and/or affected hormone rhythms interfere with regular cell function. Glucocorticoids which are highly relevant within the circadian and the immune systems should at least be mentioned.
• The cellular redox state which is controlled by the molecular clockwork and highly relevant for tumor development and progression should at least be mentioned.
• In chapter 3.2. (or 3.2), you may cite the studies of Ali et al on the role of Bmal1 in adult neurogenesis in SVZ

Deficiency of the clock gene Bmal1 affects neural progenitor cell migration. Ali AAH, Schwarz-Herzke B, Mir S, Sahlender B, Victor M, Görg B, Schmuck M, Dach K, Fritsche E, Kremer A, von Gall C. Brain Struct Funct. 2019 Jan;224(1):373-386. doi: 10.1007/s00429-018-1775-1. Epub 2018 Oct 19. PMID: 30341743

• In chapter 4.1, you may cite the study by Hassan et al. on HCC:
  Time-dependent changes in proliferation, DNA damage and clock gene expression in hepatocellular carcinoma and healthy liver of a transgenic mouse model.

• Figure 2 implies that a major problem of brain tumors derived from NSC, oligodendrocytes and astrocytes is the formation of secondary tumors. However, metastasis of glioma is a very rare event and not a major problem. In addition, the structure of the basal membrane and the capillaries is different in brain and periphery. You may break up figure 2 to illustrate the two entities brain tumors and peripheral epithelial/mesenchymal tumors (here with the major problem of metastasis including brain metastasis).  
• Page 10 lane 593: “Altered melatonin levels in the body because of excessive nocturnal light exposure to the retina or disrupted circadian timing is a likely contributor to cancer because of melatonin’s anticancer properties [75].” Please note that the C57BL/6 mouse which is most commonly used, is melatonin-deficient and, as far as I know, not more prone to tumor than melatonin-proficient mouse strains.
• In chapter 4.4., you may cite : Does timing matter in radiotherapy of hepatocellular carcinoma? An experimental study in mice. Hassan SA, Ali AAH, Sohn D, Flögel U, Jänicke RU, Korf HW, von Gall C. Cancer Med. 2021 Nov;10(21):7712-7725. doi: 10.1002/cam4.4277. Epub 2021 Sep 20. PMID: 34545699
• Chapter 5: I disagree with the conclusion that “in vitro studies may provide more satisfying answers”. They may provide more insight into the cellular mechanisms and for the role of the molecular clockwork in tumor development and progression. However, consideration of the entire circadian system is mandatory and thus only in vivo studies are suitable to decipher the connection between circadian misalignment and tumor development/progression and to make further progress in the development of cancer chronotherapy.

Minor

Page 4 lane 115: “Circadian rhythms [9].” is not a complete sentence

Page 6 lane 195: ESCs are not totipotent but pluripotent

Page 9 lane 366: “consisting of an array of key proteins and their synthe-366 sis, degradation, and gene regulation activities [52].” Is not a complete sentence

Figure 2 the legend indicates a subdivision of the figure in 2A-I. However, these subdivisions cannot be found in the scheme.

Author Response

Reviewer 3:

The review by Malik et al focusses on the role of the molecular clockwork on adult neurogenesis and glioma cancer stem cells. In addition, the role of the circadian system and the relevance of circadian misalignment on tumor development and progression as well as cancer chronotherapy are briefly discussed. The topic is very interesting and highly relevant. However, the different levels of regulation are not very clearly defined. The article would benefit greatly if the authors paid more attention to the role of systemic rhythmic factors for regular and/or rhythmic cell function.

Major

 Please indicate in the title/abstract and/or the first part of the introduction that you focus on vertebrates/mammals

• The definition of the terms “circadian clock” or “clock” is not quite clear. On page 2, the circadian clock is defined as multicellular circadian oscillators, eg in SCN or peripheral oscillator. In Figure 1, the circadian clock seems to be equal to the molecular circadian clockwork. On page 10 ( lane 418) the circadian clock seems to be equal to the circadian system: “melatonin is under circadian clock control”, similarly, page 13 lane 569: “VEGF signaling cascades and components that regulate angiogenesis are 569 under clock control”. Please clearly define the components and their effects at the molecular/cellular, multicellular and systemic level.

Response: We added clarification on how the circadian clock system is composed of clocks within clocks, and how the gears of the circadian clock mechanism should be called the “molecular clock”, lines 114-123.  We also distinguished between oscillators that merely have an identified rhythmic cell component and clocks that have demonstrated or suspected inputs and outputs conveying timing information, although these are often not well known. We only addressed subcellular effects of timing signals at some places, leaving the reader with cited papers where the details can be found.  Because of the broad range of timing signals acting on various circadian clocks we were not able to include more details at the molecular level except in some areas such as nuclear receptors that act at target genes.  The molecular effects from the signaling molecules we mentioned are often described in detail in other reviews that we cite.

• Please explain how rhythmic systemic factors such as metabolic signals, light at night and chronic stress, and/or affected hormone rhythms interfere with regular cell function. Glucocorticoids which are highly relevant within the circadian and the immune systems should at least be mentioned.

Response: We added a brief description of the role of signaling through glucorticoids and other nuclear receptors at line 104 where a useful review is cited.  We also describe TGF-beta effects on clock cells at line 106 as a possible coupling agent that, along with CORT, could involve the circadian rhythms of the immune system with the rest of the clock system.

• The cellular redox state which is controlled by the molecular clockwork and highly relevant for tumor development and progression should at least be mentioned.

Response: We mentioned it briefly at line 92. Redox potentials are important for circadian rhythms and CSCs that could be described in a more specific review.  We do direct the reader to the melatonin and cancer cell model at line 443 that is relevant to this topic.

• In chapter 3.2. (or 3.2), you may cite the studies of Ali et al on the role of Bmal1 in adult neurogenesis in SVZ

Deficiency of the clock gene Bmal1 affects neural progenitor cell migration. Ali AAH, Schwarz-Herzke B, Mir S, Sahlender B, Victor M, Görg B, Schmuck M, Dach K, Fritsche E, Kremer A, von Gall C. Brain Struct Funct. 2019 Jan;224(1):373-386. doi: 10.1007/s00429-018-1775-1. Epub 2018 Oct 19. PMID: 30341743

              Response: Thanks. Added it.

• In chapter 4.1, you may cite the study by Hassan et al. on HCC:
  Time-dependent changes in proliferation, DNA damage and clock gene expression in hepatocellular carcinoma and healthy liver of a transgenic mouse model.

Response Thanks.

• Figure 2 implies that a major problem of brain tumors derived from NSC, oligodendrocytes and astrocytes is the formation of secondary tumors. However, metastasis of glioma is a very rare event and not a major problem. In addition, the structure of the basal membrane and the capillaries is different in brain and periphery. You may break up figure 2 to illustrate the two entities brain tumors and peripheral epithelial/mesenchymal tumors (here with the major problem of metastasis including brain metastasis).

Response:  We modified the labeling of Figure 2G to indicate where rhythms are likely according to the literature.  We discuss in the body of the text how some studies indicated weak or nonexistent circadian rhythms in tumors.  We indicated that 2G refers to various tumors, some with dampened or suppressed tumors. lines 721-723.  We also added a statement that glioma metastasis is rare and that brain capillaries are different, along with a reference, lines 730-731.  We decided that breaking out gliomas in the figure as a special tumor case would create too much complexity and that the disclaimer in the figure legend is better.

• Page 10 lane 593: “Altered melatonin levels in the body because of excessive nocturnal light exposure to the retina or disrupted circadian timing is a likely contributor to cancer because of melatonin’s anticancer properties [75].” Please note that the C57BL/6 mouse which is most commonly used, is melatonin-deficient and, as far as I know, not more prone to tumor than melatonin-proficient mouse strains.

Response: Thanks for this important point to add.  We included it at lines 381-383 with additional references.  For those animals that do have functioning circadian rhythms in serum melatonin we added the model at line 443.

• In chapter 4.4., you may cite : Does timing matter in radiotherapy of hepatocellular carcinoma? An experimental study in mice. Hassan SA, Ali AAH, Sohn D, Flögel U, Jänicke RU, Korf HW, von Gall C. Cancer Med. 2021 Nov;10(21):7712-7725. doi: 10.1002/cam4.4277. Epub 2021 Sep 20. PMID: 34545699

Response: Thanks.  Added it.

• Chapter 5: I disagree with the conclusion that “in vitro studies may provide more satisfying answers”. They may provide more insight into the cellular mechanisms and for the role of the molecular clockwork in tumor development and progression. However, consideration of the entire circadian system is mandatory and thus only in vivo studies are suitable to decipher the connection between circadian misalignment and tumor development/progression and to make further progress in the development of cancer chronotherapy.

Response:  We agree that how cancer cells and stem cells function in the intact animals is of greatest importance. We added that point at lines 655-657.  Also, see our response to Major Comment 3 from Reviewer 2 above.

Minor

Page 4 lane 115: “Circadian rhythms [9].” is not a complete sentence. Corrected

Page 6 lane 195: ESCs are not totipotent but pluripotent.  Corrected

Page 9 lane 366: “consisting of an array of key proteins and their synthe-366 sis, degradation, and gene regulation activities [52].” Is not a complete sentence.  Corrected

Figure 2 the legend indicates a subdivision of the figure in 2A-I. However, these subdivisions cannot be found in the scheme.  Corrected in Figure 2.  See above.

Round 2

Reviewer 2 Report

Overall, the authors revised the draft well for publication.

Reviewer 3 Report

The manuscript has improved after revision.