Reviewer ’s report  
 
Title: c-Jun N -terminal kinase activation has a prognostic implication and is negatively 
associated with FOXO1 activation in gastric cancer  
 
 
Version: 0 Date: 22 Nov 2015  
 
 
Reviewer: Jean Winter  
 
 
Reviewer's report:  
 
Background - overall it is OK, but insufficient detail, it is not convincing regarding significance 
and does not e xplain the process of JNK/FOXO1 very effectively.  
 
* Page 4 , line 3: Is gastric cancer one of the most common cancers? I would argue this 
point. The information given in the background section has not convinced me of this. 
More evidence eg incidence data, mortality rates would be helpful to convince the reader 
of the significance of this work.  
 
* Page 4 line 8: What oncogene activation/tumor suppressor?  
 
* Page 13, line 12: Again, the info given thus far has not convinced me that more 
understanding of molecul ar pathways is going to be useful for gastric cancer therapies - 
why? What therapies are used now, are they effective/ineffective?  
 
* Page 4 line 32: Which cancers?  
 
* Page 4 line 53: Is this from humans? More detail needed here, who is "They" need 
adequate reference here  
* Page 5 line 30: What is the aim of this study? What are you trying to determine, this 
needs to be clear here. This paragraph does not clearly outline the aims of the study.  
 
Methods - Details for the WB, shRNA knockdown, Lu reporter assay, IF and CFU assay are all 
well described. However, the first few sections of the methods including the patient and tissue 
samples and immunohistochemistry of tissue microarrays (maybe also the cell culture. JNK 
inhibitor section also) needs improvement, aft er all these are the most important aspects of this 
study:  
 
* Page 5 line 50: What does "The files" mean? This is not clear  
 
* The whole section of "patient and tissue sampling" is all a little hard to follow. This 
needs more clearly explained detail, maybe a flow diagram or table of some description 
might aid in this.  
 
* Page 6 line 30: "tissue array blocks showed excellent agreement" this doesn't make sense, 
please explain this more clearly.  
 
* The reviewer suggests including more detail on the IHC reactions steps eg antigen 
retrieval etc, considering this is part of the major aspect of the study. The WB and IF had 
a lot more detail of the methods than the IHC.  
 
* Page 7 line 39 - Please show these concentrations somewhere or list them in the text.  
 
 
Results  
 
* Page 11, line 44: "We wondered…" recommend to re -word this to something less 
subjective.  
* Page 11 line 39 (and troughout the text, tables and figures) : When writing "expression" I 
would suggest including IHC or protein expression or tissue microarray to i ntroduce the 
paragraph, particularly as WB and IF methods were also included here, it gets a little 
confusing at times as to which expression you are referring to.  
 
* Page 12 line 5: "JNK activation is related to good prognosis" this is not very clear, plea se 
rephrase.  
 
* Page 14, line 46: "To provide direct evidence that the role of JN in colony formation is 
mediated by FOXO1…" These are quite strong words, I would tone this down a little…  
 
Discussion - overall I think the discussion is lacking. There is a lo t of revising the results from 
the current study, with interpretation simply made by analyzing their own data and not 
referencing any other studies. Although, I understand this is a novel study in gastric cancer, 
however, I would like to see a lot more dis cussion to include information from external sources.  
 
* Page 15 line 32: What specific clinicopathological features?  
 
* Page 15 line 37, last sentence: This is a confusing statement, needs to be reworded for 
clarity.  
 
* Page 16 line8: Do you mean hyper -proliferation (ie growth promoting activity) is an 
important process in initiation? Certainly it can be, but is proliferation truly a marker of 
neoplasic potential in gastric cancer?  
 
* Page 16 line 21: Please give full na me of pTNM in abbreviation list.  
 
* Page 17, line 48 3rd  paragraph: Is this the only limitation of this study? The reviewer 
would argue that certainly tissue microarray may not be the best method to use here and a 
full biopsy sample would be much more incl usive and indicative. Also, What about 
including some in vivo data?  
 
* Page 18, in 23: What further investigations? There needs to be more reference to how we 
might actually determine whether these "associations" actually have some true biological 
significa nce in terms of using JNK as a predictive biomarker for JNK….if this is what 
this study was aiming to do (this is not made clear in the aim/hypothesis paragraph of the 
background).  
 
Overall I think the data here is very interesting and a significant contri bution to the already 
limited data available on JNK/FOXO1 role in gastric cancer and could be the start of more data 
to prove these associations. Association may not necessarily be causal, and I think this is another 
important point to consider here.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the ma nuscript:  
 
Needs  some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees,  funding, or salary from 
an organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial com peting interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests'  
 
I agree t o the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: c-Jun N -terminal kinase activation has a prognostic implication and is negatively 
associated with FOXO1 activation in gastric cancer  
 
 
Version: 0 Date: 22 Nov 2015  
 
 
Reviewer: Jean Winter  
 
 
Reviewer's report:  
 
Background - overall it is OK, but insufficient detail, it is not convincing regarding significance 
and does not e xplain the process of JNK/FOXO1 very effectively.  
 
* Page 4 , line 3: Is gastric cancer one of the most common cancers? I would argue this 
point. The information given in the background section has not convinced me of this. 
More evidence eg incidence data, mortality rates would be helpful to convince the reader 
of the significance of this work.  
 
* Page 4 line 8: What oncogene activation/tumor suppressor?  
 
* Page 13, line 12: Again, the info given thus far has not convinced me that more 
understanding of molecul ar pathways is going to be useful for gastric cancer therapies - 
why? What therapies are used now, are they effective/ineffective?  
 
* Page 4 line 32: Which cancers?  
 
* Page 4 line 53: Is this from humans? More detail needed here, who is "They" need 
adequate reference here  
* Page 5 line 30: What is the aim of this study? What are you trying to determine, this 
needs to be clear here. This paragraph does not clearly outline the aims of the study.  
 
Methods - Details for the WB, shRNA knockdown, Lu reporter assay, IF and CFU assay are all 
well described. However, the first few sections of the methods including the patient and tissue 
samples and immunohistochemistry of tissue microarrays (maybe also the cell culture. JNK 
inhibitor section also) needs improvement, aft er all these are the most important aspects of this 
study:  
 
* Page 5 line 50: What does "The files" mean? This is not clear  
 
* The whole section of "patient and tissue sampling" is all a little hard to follow. This 
needs more clearly explained detail, maybe a flow diagram or table of some description 
might aid in this.  
 
* Page 6 line 30: "tissue array blocks showed excellent agreement" this doesn't make sense, 
please explain this more clearly.  
 
* The reviewer suggests including more detail on the IHC reactions steps eg antigen 
retrieval etc, considering this is part of the major aspect of the study. The WB and IF had 
a lot more detail of the methods than the IHC.  
 
* Page 7 line 39 - Please show these concentrations somewhere or list them in the text.  
 
 
Results  
 
* Page 11, line 44: "We wondered…" recommend to re -word this to something less 
subjective.  
* Page 11 line 39 (and troughout the text, tables and figures) : When writing "expression" I 
would suggest including IHC or protein expression or tissue microarray to i ntroduce the 
paragraph, particularly as WB and IF methods were also included here, it gets a little 
confusing at times as to which expression you are referring to.  
 
* Page 12 line 5: "JNK activation is related to good prognosis" this is not very clear, plea se 
rephrase.  
 
* Page 14, line 46: "To provide direct evidence that the role of JN in colony formation is 
mediated by FOXO1…" These are quite strong words, I would tone this down a little…  
 
Discussion - overall I think the discussion is lacking. There is a lo t of revising the results from 
the current study, with interpretation simply made by analyzing their own data and not 
referencing any other studies. Although, I understand this is a novel study in gastric cancer, 
however, I would like to see a lot more dis cussion to include information from external sources.  
 
* Page 15 line 32: What specific clinicopathological features?  
 
* Page 15 line 37, last sentence: This is a confusing statement, needs to be reworded for 
clarity.  
 
* Page 16 line8: Do you mean hyper -proliferation (ie growth promoting activity) is an 
important process in initiation? Certainly it can be, but is proliferation truly a marker of 
neoplasic potential in gastric cancer?  
 
* Page 16 line 21: Please give full na me of pTNM in abbreviation list.  
 
* Page 17, line 48 3rd  paragraph: Is this the only limitation of this study? The reviewer 
would argue that certainly tissue microarray may not be the best method to use here and a 
full biopsy sample would be much more incl usive and indicative. Also, What about 
including some in vivo data?  
 
* Page 18, in 23: What further investigations? There needs to be more reference to how we 
might actually determine whether these "associations" actually have some true biological 
significa nce in terms of using JNK as a predictive biomarker for JNK….if this is what 
this study was aiming to do (this is not made clear in the aim/hypothesis paragraph of the 
background).  
 
Overall I think the data here is very interesting and a significant contri bution to the already 
limited data available on JNK/FOXO1 role in gastric cancer and could be the start of more data 
to prove these associations. Association may not necessarily be causal, and I think this is another 
important point to consider here.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the ma nuscript:  
 
Needs  some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees,  funding, or salary from 
an organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial com peting interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests'  
 
I agree t o the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: c-Jun N -terminal kinase activation has a prognostic implication and is negatively 
associated with FOXO1 activation in gastric cancer  
 
 
Version: 0 Date: 22 Nov 2015  
 
 
Reviewer: Jean Winter  
 
 
Reviewer's report:  
 
Background - overall it is OK, but insufficient detail, it is not convincing regarding significance 
and does not e xplain the process of JNK/FOXO1 very effectively.  
 
* Page 4 , line 3: Is gastric cancer one of the most common cancers? I would argue this 
point. The information given in the background section has not convinced me of this. 
More evidence eg incidence data, mortality rates would be helpful to convince the reader 
of the significance of this work.  
 
* Page 4 line 8: What oncogene activation/tumor suppressor?  
 
* Page 13, line 12: Again, the info given thus far has not convinced me that more 
understanding of molecul ar pathways is going to be useful for gastric cancer therapies - 
why? What therapies are used now, are they effective/ineffective?  
 
* Page 4 line 32: Which cancers?  
 
* Page 4 line 53: Is this from humans? More detail needed here, who is "They" need 
adequate reference here  
* Page 5 line 30: What is the aim of this study? What are you trying to determine, this 
needs to be clear here. This paragraph does not clearly outline the aims of the study.  
 
Methods - Details for the WB, shRNA knockdown, Lu reporter assay, IF and CFU assay are all 
well described. However, the first few sections of the methods including the patient and tissue 
samples and immunohistochemistry of tissue microarrays (maybe also the cell culture. JNK 
inhibitor section also) needs improvement, aft er all these are the most important aspects of this 
study:  
 
* Page 5 line 50: What does "The files" mean? This is not clear  
 
* The whole section of "patient and tissue sampling" is all a little hard to follow. This 
needs more clearly explained detail, maybe a flow diagram or table of some description 
might aid in this.  
 
* Page 6 line 30: "tissue array blocks showed excellent agreement" this doesn't make sense, 
please explain this more clearly.  
 
* The reviewer suggests including more detail on the IHC reactions steps eg antigen 
retrieval etc, considering this is part of the major aspect of the study. The WB and IF had 
a lot more detail of the methods than the IHC.  
 
* Page 7 line 39 - Please show these concentrations somewhere or list them in the text.  
 
 
Results  
 
* Page 11, line 44: "We wondered…" recommend to re -word this to something less 
subjective.  
* Page 11 line 39 (and troughout the text, tables and figures) : When writing "expression" I 
would suggest including IHC or protein expression or tissue microarray to i ntroduce the 
paragraph, particularly as WB and IF methods were also included here, it gets a little 
confusing at times as to which expression you are referring to.  
 
* Page 12 line 5: "JNK activation is related to good prognosis" this is not very clear, plea se 
rephrase.  
 
* Page 14, line 46: "To provide direct evidence that the role of JN in colony formation is 
mediated by FOXO1…" These are quite strong words, I would tone this down a little…  
 
Discussion - overall I think the discussion is lacking. There is a lo t of revising the results from 
the current study, with interpretation simply made by analyzing their own data and not 
referencing any other studies. Although, I understand this is a novel study in gastric cancer, 
however, I would like to see a lot more dis cussion to include information from external sources.  
 
* Page 15 line 32: What specific clinicopathological features?  
 
* Page 15 line 37, last sentence: This is a confusing statement, needs to be reworded for 
clarity.  
 
* Page 16 line8: Do you mean hyper -proliferation (ie growth promoting activity) is an 
important process in initiation? Certainly it can be, but is proliferation truly a marker of 
neoplasic potential in gastric cancer?  
 
* Page 16 line 21: Please give full na me of pTNM in abbreviation list.  
 
* Page 17, line 48 3rd  paragraph: Is this the only limitation of this study? The reviewer 
would argue that certainly tissue microarray may not be the best method to use here and a 
full biopsy sample would be much more incl usive and indicative. Also, What about 
including some in vivo data?  
 
* Page 18, in 23: What further investigations? There needs to be more reference to how we 
might actually determine whether these "associations" actually have some true biological 
significa nce in terms of using JNK as a predictive biomarker for JNK….if this is what 
this study was aiming to do (this is not made clear in the aim/hypothesis paragraph of the 
background).  
 
Overall I think the data here is very interesting and a significant contri bution to the already 
limited data available on JNK/FOXO1 role in gastric cancer and could be the start of more data 
to prove these associations. Association may not necessarily be causal, and I think this is another 
important point to consider here.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the ma nuscript:  
 
Needs  some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees,  funding, or salary from 
an organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial com peting interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests'  
 
I agree t o the open peer review policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant