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  • Kohji Yamada 1,*,†,
  • Yoshito Hannya 1,2,† and
  • Kiyotsugu Yoshida 1,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Yuhua Tian

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript “Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by Cancer-Related Unconventional Protein Secretion”, authors aimed to demonstrate the role of E-Syt1 in liver cancer, especially in secretion of PKCδ. The purpose is interesting, timely and has scientific significance. The manuscript is well designed and conducted and draw convincing conclusion. My major concern is that the data are part of a previous study (doi 10.1073/pnas.2202730119), but they are fragmented in a new publication. Item 3.2 “E-Syt1 contributes to PKCδ secretion” should have been included in the previous study and not submitted in a new publication.

My recommendation: Authors should clarify the question raised before the manuscript is published.

Author Response

1, Comment: “In the manuscript “Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by Cancer-Related Unconventional Protein Secretion”, authors aimed to demonstrate the role of E-Syt1 in liver cancer, especially in secretion of PKCδ. The purpose is interesting, timely and has scientific significance. The manuscript is well designed and conducted and draw convincing conclusion. My major concern is that the data are part of a previous study (doi 10.1073/pnas.2202730119), but they are fragmented in a new publication. Item 3.2 “E-Syt1 contributes to PKCδ secretion” should have been included in the previous study and not submitted in a new publication. Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by Cancer-Related Unconventional Protein Secretion My recommendation: Authors should clarify the question raised before the manuscript is published.”

 

OUR RESPONSE: Thank you for your important remarks.  The previous PNAS paper focused mainly on the mechanism of secretion of cytosolic proteins.  The generality of E-Syt1-mediated secretion in liver cancer cell lines was not clear.  In this respect, the present study differs from previous studies in that it was used with multiple cell lines, including HuH7 cells and newly purchased HepG2 cells, as described in section 3.2, and we consider this to be an important study in that we could confirm the reproducibility of this phenomenon.  The phenomenon itself is novel and it is important to be easily reproducible in the literature.  The point of this research is that E-Syt1, an important factor in the CUPS system, is involved in tumorigenesis, and we consider it possible to apply nucleic acid therapy and genome therapy in the future by studying whether it is a driver gene or not.

In summary, this study is important in cancer research in that it discusses whether E-Syt1 contributes to tumorigenesis, which is different from the previous paper, which focuses on the basic cell biology of secretion mechanisms.  Note that the item 3.2, "E-Syt1 contributes to PKCd secretion," was added as "in liver cancer cell lines," since multiple cell lines were used.

 

We have added the following phrase to accompany the addition of main text.

Page 5, line 202; “3.2. E-Syt1 contributes PKCd secretion in liver cancer cell lines”

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this research article, the author demonstrated that extended synaptotagmin 1 plays a critical role in liver cancer progression, mediated by the unconventional secretion of cytosolic proteins, employing various molecular biology techniques. Overall, the article is well-organized and systematically structured. However, several concerns arise in the results section:

  1. In Figure 2D (left panel), the difference between the control and E-syt1 knockout (KO) groups is not readily discernible in the PLA results. The author should consider substituting this image with one that shows a more significant difference between the groups.
  2. In Figure 5C, the author only reported the values of the hazard ratio and log-rank statistics. It would be beneficial for reader’s comprehension if the significance and implications of these two parameters were clarified.
  3. The resolution of all figures is insufficient for publication, particularly in comparison to the higher-quality figures in the author’s previous PNAS publication on a similar topic.

Author Response

July 28, 2025

MS ID: molecules-2395672

MS TITLE: Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by Cancer-Related Unconventional Protein Secretion

 

Dear Dr. Jim Zhu,

We sincerely appreciate the opportunity to submit a revised version of our manuscript for your consideration.  We would also like to thank you as well as the reviewers for your critique and valuable comments.  The revised manuscript has been attached with changes marked in the text along with a point-by-point response to each of the reviewers’ comments provided below.

 

RESPONSE TO REVIEWER COMMENTS:

REVIEWER COMMENTS ARE IN ITALICS

OUR RESPONSES FOLLOW AND ARE IN NORMAL FONT (IN SOME CASES, PHRASES THAT SHOULD BE EMPHASIZED ARE IN BOLD)

 

 

 

Reviewer #3

(Remarks to the Author)

Comment: “In Figure 2D (left panel), the difference between the control and E-syt1 knockout (KO) groups is not readily discernible in the PLA results. The author should consider substituting this image with one that shows a more significant difference between the groups.”

 

 

OUR RESPONSE: Thank you for your important remarks.  As you mention, it is difficult to express show the difference in appearance in the PLA staining.  Therefore, in addition to conducting quantitative measurements, we have posted objective average images, and we consider that this is the most scientifically fair approach.

Comment: “In Figure 5C, the author only reported the values of the hazard ratio and log-rank statistics. It would be beneficial for reader’s comprehension if the significance and implications of these two parameters were clarified.”

 

 

OUR RESPONSE: Thank you for your important comment.  In many cases of cancer researches, the levels of gene expression has correlated to poor prognosis.  Therefore, as you mention, we should add the sentence to main, “, indicating that E-Syt1 expression effected on liver cancer”

 

We have added the following phrase to accompany the addition of main text.

Page 9, line 6 “, indicating that E-Syt1 expression effected on liver cancer”

 

 

 

Comment: “The resolution of all figures is insufficient for publication, particularly in comparison to the higher-quality figures in the author’s previous PNAS publication on a similar topic.”

 

OUR RESPONSE: Thank you for your comment.  As you mention, I exchanged the new high quality figures (1 to 5).