Atypical contribution of caspase-3 to melanoma cancer cell motility by regulation of coronin 1B activity

  1. Kevin Berthenet
  2. Serena Diazzi
  3. Catherine Jamard
  4. Kinga Stopa
  5. Stefan Dragan
  6. Deborah Fanfone
  7. Trang Nguyen
  8. Nathalie Al
  9. Francois Virard
  10. Olivier Meurette
  11. Nikolay Popgeorgiev
  12. Hector Hernandez-Vargas
  13. Julien Ablain
  14. Gabriel Ichim

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Abstract

Recent studies have unveiled unexpected connections between cell death and cell motility. While traditionally recognized for their pro-apoptotic roles, caspases have emerged as regulators of physiological processes beyond cell death, including cellular differentiation and motility. In some particularly aggressive cancers like melanoma, caspase-3, a prominent executioner caspase, is unexpectedly and inexplicably highly expressed. Here, we describe a novel non-apoptotic role for caspase-3 in melanoma cell motility. Through comprehensive molecular and cellular analyses, we demonstrate that caspase-3 is constitutively associated with the cytoskeleton and crucially regulates melanoma cell migration and invasion in vitro and in vivo . Mechanistically, caspase-3 interacts with and modulates the activity of coronin 1B, a key regulator of actin polymerization, thereby promoting melanoma cell motility, independently of its apoptotic protease function. Furthermore, we identify specificity protein 1 (SP1) as a transcriptional regulator of CASP3 expression, and show that its inhibition reduces caspase-3 expression and impairs melanoma cell migration. Overall, this study provides insights into the multifaceted roles of caspase-3 in cancer progression, highlighting its relevance as a novel target for anti-metastatic therapies.

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  1. Review Commons

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    Reply to the reviewers

    Manuscript number: RC-2024-02648

    Corresponding author(s): Kevin Berthenet (kevin.berthenet@lyon.unicancer.fr) and Gabriel Ichim (gabriel.ichim@lyon.unicancer.fr)

    1. General Statements

    We thank all the reviewers for their time and their constructive criticism, based on which we propose the revision plan detailed bellow. All our responses are indicated in italics font. When is the case, the figures for the reviewers are included just below the answer. Only where indicated they have been included in the manuscript. The line numbers indicated here refer to those in original manuscript.

    The two reviews are listed in full at the end of the document.

    2. Description of the planned revisions

    Reviewer #1

    In this manuscript, the authors report a non-apoptotic role for caspase 3 in promoting cell migration. RNA sequencing revealed a "gene signature" associated with caspase 3 knockdown in a melanoma cell line, although there is no investigation of the connection between caspase 3 expression and the regulation of gene expression. Mass spectrometry-based experiments (AP-MS and BioID) identified numerous interacting proteins, with coronin 1B being the most extensively characterized. Data provided indicates that there is a direct interaction between caspase 3 and coronin 1B, and that caspase 3 influences coronin 1B phosphorylation basally and following ligand stimulation. Both proteins are required for efficient cell migration in scratch wound assays. Data is provided indicating that the actions of caspase 3 are independent of proteolytic activity, although the pharmacological inhibition of caspase activity is not complete, nor is the knockdown of BAX/BAK, making these conclusions poorly substantiated. Evaluation of pathways regulating caspase 3 expression implicates the SP1 transcription factor.

    Response: We thank the reviewer for their supportive comment. Regarding specific pharmacological inhibition of caspase-3, work is under way to complement the results obtained with a pan-caspase inhibitor (qVD-OPh). We will use specific effector caspases inhibitors, complemented by several other approaches: complete KO of BAX and BAK proteins to prevent all eventual mitochondrial permeabilization and low-level effector caspase activation, overexpression (OE) of the anti-apoptotic protein BCL-xL to also prevent residual mitochondrial permeabilization, while also OE XIAP, a potent caspase inhibitor. The promising preliminary data using two effector caspases specific inhibitors (Ac-DEVD-CHO and Ac-DNLD-CHO) in two different melanoma cells, during wound healing migration, is shown below, with no effect on melanoma cell migration.

    Line 129 - The data in Sup. Fig. 1H-L are technical, but where are the mass spectrometry results from the BioID2 experiments? These technical figures are really only relevant if the BioID2 system has been used for protein pull-downs, not for the IF analysis in Fig. 2B.

    Response: We apologize for lack of precision in the article logical flow, we will now incorporate the MS data based on the BioID2 experiment earlier in the manuscript.

    Line 143 - Figure 2C - it is not entirely convincing that caspase 7 is not associated with the cytoskeleton, there is a visible band in lysates from both cell lines, in contrast with GAPDH which is convincingly cytoplasmic. This is particularly true in the WM852 cell lines, in which the Caspase 3 band is almost the same as Caspase 7. These results would also be more convincing if there was IF of Caspase 7 and actin to show whether it is or is not enriched in regions of higher F-actin levels.

    Response: Indeed, our data points towards an enrichment of caspase-3 at the cell cortex. Since generally caspase-7 protein levels are lower, we detected it less in the cytosolic fraction. As suggested, now we performed more sensitive IF colocalization confocal imaging between caspase-7 and F-actin and find it also partially localized to the cortical cytoskeleton (see below). However, this effector caspase is not involved in melanoma cell migration (see wound healing assay below, with two different siRNAs for CASP7 and the positive control of siRNA CASP3).

    Figure 2D - knockdowns with only a single siRNA are insufficient, this should be replicated with additional siRNAs. In addition to the effect on actin anisotropy, it appears as though cells are smaller, is this and any other morphological changes reproducible?

    Response: We plan to strengthen the data shown in Fig.2D with additional siRNAs, as shown below. In addition, high-content screening (HCS) microscopy will provide several other cell morphology descriptors.

    Figure 2D-E. Is it cytochalasin B or D used in these experiments? The text and figures don't agree with each other.

    1. Figure 2F-G, same comments above for 2D-E (i.e. comments 3 & 4).

    Response: The experimental conditions will be better detailed in the revised manuscript.

    Figure 2F-G, it appears as though the fewer focal adhesions in the Caspase 3 knockdown cells are bigger per focal adhesion, is this a consistent result? If so, what is the explanation?

    Response: In addition to number, we also plan to quantify the size of focal adhesions.

    Figure 2H - it's not clear how this RNAseq data is relevant to the manuscript. There are some genes in the heat map, but it's not clear which ones are changed in their expression in the caspase 3 knockdown cells, nor is it clear how this is relevant to the proposed mechanisms of Caspase 3 interacting with and influencing the phosphorylation of coronin 1B. If there is no connection, then these data can be removed.* *

    Response: As suggested by the reviewer, the RNAseq data presented in Figure 2H will be removed from the revised manuscript since it is not very relevant.

    Supp. Figure 3 - given that there is data from multiple siRNAs for the incucyte migration data, it should be in the primary figures. And since there are multiple siRNAs and CRISPR/Cas9 KO cells, there should be nothing limiting the replication of the other data presented from only a single siRNA.

    Response: Several siRNA are now used for replicating key results as shown above.

    Figure 3A - how was cell adhesion measured? The methods section says "cell adhesion was determined through cell shape analysis and scoring" But this is very vague.

    Response: We thank the reviewer for spotting out this ambiguity, in the revised manuscript we will be more precise in Material and Methods section.

    Figure 3L - was the Casp7 knockdown experiments done with multiple siRNAs? Both melanoma cell lines? Why is this figure only shown out to 24 hours, whereas the other Incucyte experiment run out to 48 hours? Where is the western blot confirming the caspase 7 knockdown? This is important to establish a clear lack of effect.

    Response*: We apologize for lacking more details, we now provide several siRNA for caspase-7, all showing no or minimal effect of melanoma cell migration (see answer to point 2). *

    Line 190 - it is not true to say that in the presence of QVD there is no longer any caspase activity induced by actinomycin D/ABT263 in supplemental Figures 3J-K. The way that the Y axis has been broken diminishes the difference between untreated and treated cells. In fact, there is apparently over 3-4 times more caspase activity in the actinomycin D/ABT263 treated cells in the presence of QVD relative to basal caspase activity. As a result, it cannot be concluded that there is no residual caspase activity.

    Response: We were not precise enough in describing the data in S3J-K. In the revised manuscript we will clearly say that since treatment with a pan-caspase inhibitor does not have the effect of lowering any basal caspase activity (column 1 versus 2), we conclude that in melanoma cells (WM793 and WM852) there is no basal caspase activation that could drive cell motility. The ActD/ABT263 treatment was used as positive control for bona fide induction of effector caspase activation. These results will be complemented by BAX/BAK DKO and BCLxL OE.

    Line 192 - Does the knockout of BAX/BAK (which apparently reduced but did not eliminate BAX/BAK protein levels in Supp. Fig. 3L) actually "completely block" caspase activity via the mitochondrial pathway? This has not been demonstrated.

    Response: We now provide a fluorometric effector caspases assay showing abrogation of caspase activity in BAX/BAK DKO cells (see below, caspase activating treatment is ActinomycinD plus ABT263). In addition, we will improve the DKO efficacy.

    Line 217 - coronin 1B was a hit from which assays? IP-MS and/or BioID2? I see that this is shown in Figure 5A but not referenced in this sentence.

    Line 218 - the reference to Figure 5A should be in the previous sentence. Line 220 - Can it really be said that the interaction is specific since there is a coronin 1B band in the GFP "negative" control?__ __

    Response*: The revised manuscript will address these inadequacies. *

    Line 222 - it is a good control to show that siRNA-knockdown of Caspase 3 reduced the PLA signal in Figure 5C, but the reciprocal experiment of looking at what happens with Coronin 1B knockdown should be included. How does the PLA signal relate to phalloidin-stained F-actin?

    Response: The proximity ligation assay (PLA) is now complemented by KD of Coronin 1B (see below) and we will try to also add the phalloidin staining for F-actin, if compatible with the PLA protocol.

    Line 224 - looking at the line scans, is the lack of recruitment of coronin 1B to the F-actin at the edge of the protrusion in the Caspase 3 knockdown cells reproducible? Is the point that caspase 3 recruits Coronin 1B? There is an obvious difference in the F-actin at the cell edge, but if the F-actin were as dense in the Caspase 3 knockdown cells as they are for the control, would the same lack of coronin 1B be apparent?

    Response: This aspect will be better addressed/discussed in the revised manuscript.

    Line 227 - where is the western blot showing the effectiveness of the coronin 1B knockdown to accompany Figure 5F.

    Response: The efficacy of coronin 1B KD will be added in the revised manuscript.

    Figure 5G - the blots indicate that there is no change in phospho-PKCalpha in the caspase 3 knockdown cells, although phospho-coronin 1B does decrease. This has not been commented upon in the text. Is the implication that there is a non-PKCalpha mediated mechanism for coronin 1B phosphorylation that is dependent on caspase 3?

    Figure 5H - following from the previous point, there is no phospho-PKCalpha blot that would be a positive control for the effect of PDGF stimulation on PKC activation, in control and caspase 3 knockdown cells, to evaluate whether the effect on coronin 1B phosphorylation was upstream or downstream of PKCalpha. This is also true for Supp. Fig. 4H.

    Response*: Since there are several PKC isoforms that might be co-expressed in melanoma cells, it is possible that PKCalpha is not the one responsible for phosphorylating Coronin 1B. We will be more precise in our investigations by using a pan-phospho-PKC antibody. *

    Does phosphorylation of coronin 1B affect its interaction with caspase 3?

    Response: We will assess by Co-IP the interaction of caspase-3 with both non-phosphorylated and phosphorylated Coronin 1B.

    Figure 6 - as before, only a single siRNA to knockdown SP1 is insufficient to robustly support the conclusions.

    Response: As shown below, we addressed this helpful comment by using several siRNAs to assess the role of SP1 in melanoma cell motility, in two different melanoma cell lines.

    Reviewer #2

    In this manuscript, the authors provide substantial amounts of experimental evidence that caspase-3, more precisely pro-caspase-3, might be involved in promoting melanoma cell migration and invasion. As such, this function, which might stem from scaffolding roles independent of proteolytic activity (yet not shown entirely convincingly), could possibly be similar to those attributed to other caspases, yet the latter omitted experiments testing for the necessity of enzyme activity. The data are novel and interesting and obviously deserve publication. Yet, a number of criticisms need to be listed.

    Response*: We thank the reviewer for upholding the novelty of our study. As also rightfully pointed by R1, we will strive in a revised manuscript to definitely show that caspase-3 participate to melanoma cell motility independently of its pro-apoptotic protease role: we will use two effector caspases specific inhibitors (Ac-DEVD-CHO and Ac-DNLD-CHO, as shown above) complemented by several other approaches: complete KO of BAX and BAK protein to prevent all eventual mitochondrial permeabilization and low-level effector caspase activation, OE of the anti-apoptotic protein BCL-xL to also prevent residual mitochondrial permeabilization, while also OE XIAP, a potent caspase inhibitor. *

    • First and foremost, I don't seem to find ethical approval information on the animal experiments. While I do not work with zebrafish myself, I am also somewhat concerned by the size of tumours seen in some of the depicted fish. It is highly important that appropriate information in this direction, including possible endpoints, is provided. Response*: We completely agree with the reviewer, yet the ethical approval is already provided in the manuscript (line 588) and will be complemented by adding the endpoints. *

    The second major issue lies in figure 1. The figure as a whole seems to be very much forced to support or motivate later experimental findings. The authors lack sufficient clarity on some of the approaches and seem to judge on the data to a good bit as they see fit. (…)

    I´d suggest to largely take out Fig1 in its current form, spend time on properly describing any analysis of public data, carefully interpret these and move them probably to the end of the results. Currently, it just leaves the impression that the data were pushed as hard as possible to promote the good work that follows.

    Response*: We will carefully consider the reviewer’s comments and rework the bioinformatics analysis presented in Figure 1 (and associated supplementary figure), making sure we will present certain data as correlation (and not causality) and go into more details on the physio-pathological features of melanoma patients with low/high caspase-3 expression. *

    The text on line 129ff seems to have omitted any outcomes from the Suppl. Fig1H-L. What was found and what are we supposed to learn from this?

    Response: We apologize for lack of precision in the article logical flow, we will now incorporate the MS data based on the BioID2 experiment earlier in the manuscript.* *

    Lines 146/147 state similar effects upon CASP3 depletion and cytochalasin D. I cannot make that out from Fig.2D. Can you be more specific or visualize this better?

    Response: We will fix this by including zoomed and detailed images of individual cells.

    • Is it possible to state whether effects such as in Fig.3B are general rather than showing just 1 cell?

    Response: The defects in cell adhesion for caspase-3-depleted cells are quantified in Figure 3A. Moreover, we will add representative images.

    It is unclear how the genes in Fig.2H were defined and why would all of these differ (unless this was an inclusion criterion for the panel). Are these considered to be downstream of CASP3 somehow? I don't fully get the message here. Is this panel even required here?

    Response: As it brings little information, panel 2H will be excluded from the revised manuscript.

    To fully prove independence of caspase-3 activity, it would be appropriate to k/o caspase-3 to then reconstitute the cells with inactive caspase-3.

    Response: We will try our best of addressing this comment in the revised manuscript.

    Fig.4C and associated text: Statements on changes in tumor size cannot be made from data on tumor free survival.

    Response: We apologize for the misleading data interpretation; this will be tuned down in a revised manuscript.

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors provide substantial amounts of experimental evidence that caspase-3, more precisely pro-caspase-3, might be involved in promoting melanoma cell migration and invasion. As such, this function, which might stem from scaffolding roles independent of proteolytic activity (yet not shown entirely convincingly), could possibly be similar to those attributed to other caspases, yet the latter omitted experiments testing for the necessity of enzyme activity. The data are novel and interesting and obviously deserve publication. Yet, a number of criticisms need to be listed.

    Major comments

    • First and foremost, I don't seem to find ethical approval information on the animal experiments. While I do not work with zebrafish myself, I am also somewhat concerned by the size of tumours seen in some of the depicted fish. It is highly important that appropriate information in this direction, including possible endpoints, is provided.
    • The second major issue lies in figure 1. The figure as a whole seems to be very much forced to support or motivate later experimental findings. The authors lack sufficient clarity on some of the approaches and seem to judge on the data to a good bit as they see fit. For example
      • The authors claim CASP3 expression is high in skin, yet the data in Fig.1A proofs this to be wrong, since it is rather medium across the range of available tissues.
      • The authors state that CASP3 stood out to be highly expressed in primary and metastatic melanoma cells, but don't state against what they compared. Other caspases or non-melanoma cell lines? The latter would be relevant comparison, I suppose. Also, names of cell lines were omitted in the figure as were information on whether they were from primary or metastatic tissue.
      • The authors state that CASP3 expression is "clinically relevant" since it differed between primary and metastatic classified TCGA cases. Why would that have clinical relevance at all? It simply correlates with staging. Clinical relevance for example could be found in data where e.g. CASP3 expression in primary melanoma associates with higher risk for recurrence or progression to metastasis.
      • The associated supplemental fig. 1 needs to be criticised as well. Panel S1D shows a Kaplan Meyer plot of high UP vs low UP signatures. Looking at the survival times plotted (less than one year), one wonders how this plot was generated. What stages were included and why, were these balanced in size, what are the group sizes, and why was a cutoff of 300 days chosen (I can only guess this might have been limited to late stage disease, but what would be the point?). There doesn´t seem to be any useful information included in this to make this somewhat interpretable from the disease or clinical side, let alone usefulness when taking into account other confounding variables. Please also note that the TCGA data need to be looked at with great care, since dates of diagnosis often are not the dates when the analysed samples were taken (e.g. diagnosis might be been primary melanoma at the time the initial lesion was removed by a dermatologist, yet the associated TCGA sample was taken much later when the disease had recurred and could already be metastatic).
      • Combining the problems mentioned above with the descriptive rest of the figure leads into a vastly exaggerated but probably wished for claim that "CASP3 expression must confer melanoma cells with certain advantages, likely unrelated to the role of caspase-3 in apoptosis). I´d suggest to largely take out Fig1 in its current form, spend time on properly describing any analysis of public data, carefully interpret these and move them probably to the end of the results. Currently, it just leaves the impression that the data were pushed as hard as possible to promote the good work that follows.
    • The text on line 129ff seems to have omitted any outcomes from the Suppl. Fig1H-L. What was found and what are we supposed to learn from this?
    • Lines 146/147 state similar effects upon CASP3 depletion and cytochalasin D. I cannot make that out from Fig.2D. Can you be more specific or visualize this better?
    • Is it possible to state whether effects such as in Fig.3B are general rather than showing just 1 cell?
    • The micrographs, especially those that were quantitatively analysed, in print display seem largely overexposed. It wouldn't make sense to measure correlations across areas that seemingly are just saturated. If the analyses were done on non-saturated raw images with higher dynamic range, please state so clearly and maybe adjust the settings more appropriately for display items.
    • It is unclear how the genes in Fig.2H were defined and why would all of these differ (unless this was an inclusion criterion for the panel). Are these considered to be downstream of CASP3 somehow? I don't fully get the message here. Is this panel even required here?
    • To fully prove independence of caspase-3 activity, it would be appropriate to k/o caspase-3 to then reconstitute the cells with inactive caspase-3.
    • Fig.4C and associated text: Statements on changes in tumor size cannot be made from data on tumor free survival.

    Minor comments:

    • Please check the sentence on line 43. Obviously this applies to living, not dead organisms, and obviously dead cells don´t migrate. Maybe simply delete.
    • Please add mol. weight markers to all panels.
    • Please check the entire manuscript to ensure, also for interpretability, that procaspase-3 and processed or active caspase-3 variants are appropriately referred to

    Significance

    Significance

    Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested.

    The following aspects are important: General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed?

    The main finding is of high significance and already supported very well by experimental evidence. The authors discuss the limitations of their study appropriately, e.g. the possibility that more advanced in vivo settings might provide additional evidence for a pro-migratory role of caspase-3. However, I would clearly NOT suggest to include e.g. mouse models in the study; in my opinions very little would be learned from that in addition to what the authors already show in a well established melanoma zebrafish model. As stated in the previous section, I am clearly very unconvinced about the first figure centering on public data repositories and their analysis. This indeed is the weakest part of the paper.

    Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...).

    Non-death roles of proteins classically linked to cell death processes are now slowly becoming appreciated more widely. As such, the contribution of the authors is very timely and noteworthy. No other convincing studies exist that would ascribe a non-proteolytic role of caspase-3 to migration or invasion. The novelty thus is high. The advance is primarily seen in the idenfication of this role and the mechanistic and functional basis of it.

    Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

    Primarily, basic researchers with links to cell death /survival regulation will appreciate these results very highly. This could be a fairly large audience. Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Cell death regulation, cancer, systems biology, cellular imaging

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, the authors report a non-apoptotic role for caspase 3 in promoting cell migration. RNA sequencing revealed a "gene signature" associated with caspase 3 knockdown in a melanoma cell line, although there is no investigation of the connection between caspase 3 expression and the regulation of gene expression. Mass spectrometry-based experiments (AP-MS and BioID) identified numerous interacting proteins, with coronin 1B being the most extensively characterized. Data provided indicates that there is a direct interaction between caspase 3 and coronin 1B, and that caspase 3 influences coronin 1B phosphorylation basally and following ligand stimulation. Both proteins are required for efficient cell migration in scratch wound assays. Data is provided indicating that the actions of caspase 3 are independent of proteolytic activity, although the pharmacological inhibition of caspase activity is not complete, nor is the knockdown of BAX/BAK, making these conclusions poorly substantiated. Evaluation of pathways regulating caspase 3 expression implicates the SP1 transcription factor.

    Major comments:

    1. Line 129 - The data in Sup. Fig. 1H-L are technical, but where are the mass spectrometry results from the BioID2 experiments? These technical figures are really only relevant if the BioID2 system has been used for protein pull-downs, not for the IF analysis in Fig. 2B.
    2. Line 143 - Figure 2C - it is not entirely convincing that caspase 7 is not associated with the cytoskeleton, there is a visible band in lysates from both cell lines, in contrast with GAPDH which is convincingly cytoplasmic. This is particularly true in the WM852 cell lines, in which the Caspase 3 band is almost the same as Caspase 7. These results would also be more convincing if there was IF of Caspase 7 and actin to show whether it is or is not enriched in regions of higher F-actin levels.
    3. Figure 2D - knockdowns with only a single siRNA are insufficient, this should be replicated with additional siRNAs. In addition to the effect on actin anisotropy, it appears as though cells are smaller, is this and any other morphological changes reproducible?
    4. Figure 2D-E. Is it cytochalasin B or D used in these experiments? The text and figures don't agree with each other.
    5. Figure 2F-G, same comments above for 2D-E (i.e. comments 3 & 4).
    6. Figure 2F-G, it appears as though the fewer focal adhesions in the Caspase 3 knockdown cells are bigger per focal adhesion, is this a consistent result? If so, what is the explanation?
    7. Figure 2H - it's not clear how this RNAseq data is relevant to the manuscript. There are some genes in the heat map, but it's not clear which ones are changed in their expression in the caspase 3 knockdown cells, nor is it clear how this is relevant to the proposed mechanisms of Caspase 3 interacting with and influencing the phosphorylation of coronin 1B. If there is no connection, then these data can be removed.
    8. Supp. Figure 3 - given that there is data from multiple siRNAs for the incucyte migration data, it should be in the primary figures. And since there are multiple siRNAs and CRISPR/Cas9 KO cells, there should be nothing limiting the replication of the other data presented from only a single siRNA.
    9. Figure 3A - how was cell adhesion measured? The methods section says "cell adhesion was determined through cell shape analysis and scoring" But this is very vague.
    10. Figure 3L - was the Casp7 knockdown experiments done with multiple siRNAs? Both melanoma cell lines? Why is this figure only shown out to 24 hours, whereas the other Incucyte experiment run out to 48 hours? Where is the western blot confirming the caspase 7 knockdown? This is important to establish a clear lack of effect.
    11. Line 190 - it is not true to say that in the presence of QVD there is no longer any caspase activity induced by actinomycin D/ABT263 in supplemental Figures 3J-K. The way that the Y axis has been broken diminishes the difference between untreated and treated cells. In fact, there is apparently over 3-4 times more caspase activity in the actinomycin D/ABT263 treated cells in the presence of QVD relative to basal caspase activity. As a result, it cannot be concluded that there is no residual caspase activity.
    12. Line 192 - Does the knockout of BAX/BAK (which apparently reduced but did not eliminate BAX/BAK protein levels in Supp. Fig. 3L) actually "completely block" caspase activity via the mitochondrial pathway? This has not been demonstrated.
    13. Line 217 - coronin 1B was a hit from which assays? IP-MS and/or BioID2? I see that this is shown in Figure 5A but not referenced in this sentence.
    14. Line 218 - the reference to Figure 5A should be in the previous sentence.
    15. Line 220 - Can it really be said that the interaction is specific since there is a coronin 1B band in the GFP "negative" control?
    16. Line 222 - it is a good control to show that siRNA-knockdown of Caspase 3 reduced the PLA signal in Figure 5C, but the reciprocal experiment of looking at what happens with Coronin 1B knockdown should be included. How does the PLA signal relate to phalloidin-stained F-actin?
    17. Line 224 - looking at the line scans, is the lack of recruitment of coronin 1B to the F-actin at the edge of the protrusion in the Caspase 3 knockdown cells reproducible? Is the point that caspase 3 recruits Coronin 1B? There is an obvious difference in the F-actin at the cell edge, but if the F-actin were as dense in the Caspase 3 knockdown cells as they are for the control, would the same lack of coronin 1B be apparent?
    18. Line 227 - where is the western blot showing the effectiveness of the coronin 1B knockdown to accompany Figure 5F?
    19. Figure 5G - the blots indicate that there is no change in phospho-PKCalpha in the caspase 3 knockdown cells, although phospho-coronin 1B does decrease. This has not been commented upon in the text. Is the implication that there is a non-PKCalpha mediated mechanism for coronin 1B phosphorylation that is dependent on caspase 3?
    20. Figure 5H - following from the previous point, there is no phospho-PKCalpha blot that would be a positive control for the effect of PDGF stimulation on PKC activation, in control and caspase 3 knockdown cells, to evaluate whether the effect on coronin 1B phosphorylation was upstream or downstream of PKCalpha. This is also true for Supp. Fig. 4H.
    21. Does phosphorylation of coronin 1B affect its interaction with caspase 3?
    22. Figure 6 - as before, only a single siRNA to knockdown SP1 is insufficient to robustly support the conclusions.

    Minor comments:

    1. Figure 2C - all caps for CASP7
    2. Figures 2D,F - Cytochalsin
    3. Figure 2H, the labelling of gene names is too small to read.
    4. Supplemental Fig 1A - why is A375 here? Why plot a graph and not just write a percentage protein remaining under the figure? There are no errors indicated, so presumably this is N = 1.
    5. Line 127 - smal

    Significance

    The manuscript is interesting and novel, making it relevant for a broad basic research audience. The role of caspase 3 in non-apoptotic biological processes is not extensively characterized, making this study an advance in the field. The methods are appropriate and well-executed. The statistical methods are mostly appropriate, although some assays (e.g. wound healing assays) do not have associated statistical analysis. Most of the conclusions are adequately substantiated by the results, but as indicated above and in the points below, this is not entirely consistent. There is an issue with only a single siRNA being used in several experiments that should be addressed.

  4. Version published to 10.1101/2024.06.27.601010v1 on bioRxiv