Cosuppression of NF-κB and AICDA Overcomes Acquired EGFR-TKI Resistance in Non-Small Cell Lung Cancer

Round 1

Reviewer 1 Report

Now this manuscript is in better form to be accepted.

Author Response

Thank you for reviewing our manuscript and for the reviewers’ comments

Author Response File: Author Response.docx

Reviewer 2 Report

The authors conclude that the treatment with an EGFR-TKI plus cosuppression of NF-κB and AICDA may be a promising strategy to overcome EGFR-TKI resistance in LACs. In my view, this manuscript in present stage could not be accepted for publication in MDPI, Cancers.

Listed below are some comments:

1. Most sentences contain grammatical and/or spelling mistakes, and the English of the manuscript must be improved before resubmission. It means that the authors must have the manuscript edited by someone competent in the English language before submission.

 

2. There seems no connection between different parts. The authors need revise the paper to connect things together to answer their research questions. 

Author Response

Thank you for reviewing our manuscript and for the reviewers’ comments. 

Reviewer 3 Report

Manuscript looks better in the present format to be accepted.

Author Response

Thank you for reviewing our manuscript and for the reviewers’ comments. 

Author Response File: Author Response.pdf

Reviewer 4 Report

Thank the authors revised the manuscript and releasing more information, especially the results from the patients. Even though the current study only includes one cell line and its resistant form, and without in vivo experiments, the patients’ data should provide more credit. I’d highly recommend the authors consider the following comments.

1.      Line 151 and Figures: a) The authors only list “One-way ANOVA” in the statistical analysis section but did not mention where it had been used. b) Please don’t just list “nonlinear regression analysis” in Figure 1,2,3 legends. Please specify if the method has been used. c) Please make sure the images in the main context are high resolution and clear to read.

2.      Line 259 Patients’ IHC results from Table S3 and figure: a) I’d suggest the authors take this as the major results that the authors should well analyze and include in the main context; b) I’d suggest the authors include some more patients’ IHC images in the main context instead of only NF-κB and provide as much as patients’ IHC images as supplementary; c) If the authors could perform at least some extra western blotting with patients’ samples to support IHC results that would be great.

 

3.      Please homogenous the format throughout the manuscript including the supplementary file.

Author Response

Thank you for reviewing our manuscript and for the comments. All the comments are valuable and very helpful for revising and improving our paper. We have revised the manuscript according to the comments and suggestions. Please see the point by point responses to the comments as listed below

Replies to Reviewer

1. Line 151 and Figures: a) The authors only list “One-way ANOVA” in the statistical analysis section but did not mention where it had been used. b) Please don’t just list “nonlinear regression analysis” in Figure 1,2,3 legends. Please specify if the method has been used.

Response 1:

Thanks for the corrections. We used only two-way student T test for statistical analysis. We edited manuscript according your correction in the method section (in lines 150-151) and figure legends 1-3 (lines 184-185, 211-212, 234-235).  

 

1. Line 151 and Figures: c) Please make sure the images in the main context are high resolution and clear to read.

Response 2:

Thanks for the suggestion. We have edited the images (Fig1-4) in the main context according to the comment.

 

2. Line 259 Patients’ IHC results from Table S3 and figure:a) I’d suggest the authors take this as the major results that the authors should well analyze and include in the main context;

Response 3:

Thanks for the suggestion. We have edited overall manuscript in the results of IHC studies in lines 239-253 and 318 according to your suggestions.

 

2. Line 259 Patients’ IHC results from Table S3 and figure:b) I’d suggest the authors include some more patients’ IHC images in the main context instead of only NF-κB and provide as much as patients’ IHC images as supplementary;

Response 4:

According to your suggestions, we replaced the image in “Figure 4” with the figure which contains (1) IHC images of the antibodies showing differential staining intensities by patient groups (Fig. 4A) (2) bar chart of H-score by patient groups (Fig 4B).  Also, supplementary Fig. 3 was modified with other images of antibodies which were not included in the main manuscript.

2. Line 259 Patients’ IHC results from Table S3 and figure: c) If the authors could perform at least some extra western blotting with patients’ samples to support IHC results that would be great.

Response 5:

Thanks for the suggestion. We tried, but most samples were not sufficient for further experiments.

 

3. Please homogenous the format throughout the manuscript including the supplementary file.

Response 6:

Thanks for the suggestion. We edited the format throughout the manuscript and especially supplementary file according to your suggestions. 

 

Yours sincerely,

Chaeuk Chung., M.D., Ph.D.; Chungnam National University School of Medicine, Munwha-ro 266, Jung-gu, Daejeon, 35015, Republic of Korea. [email protected]

Go Eun Bae M.D., Ph.D.; Chungnam National University School of Medicine, Munwha-ro 266, Jung-gu, Daejeon, 35015, Republic of Korea. Tel: +82-42-280-7797, Fax: +82-42-280-7189, e-mail: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Thank you for the updates. I do not have any further concerns now.  Good luck.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript titled “Cosuppression of NFκB and AICDA overcomes acquired EGFR-TKI resistance in non-small cell lung cancer” describes the authors tested the expression changes of signaling pathway factors in the PC9 cell line and its mutation strain by using western blot. However, the results with only some general examinations could not support the authors’ conclusion. Anyway, the followings are some concerns and comments have been pointed out that the authors may want to consider.

Concerns and Comments:

1. Line 69 Materials and Methods section: The more detailed reagents information should be provided. The dilution ratio of antibodies Supplementary Table should be cited here.
2. Line 81: Update the protein amount. The unit is not right.
3. Line 96: The “-ΔΔCT” should be superscript.
4. Lines 99-101: I’d suggest the authors use a table to present the primers. Or present them as the shRNA sequence, lines 110-115 for easier reading.
5. Lines 150-151: Please use italic p as it refers to a p-value. Please check throughout the manuscript.
6. Line 177: Figure 1A the label should be “NF-κB(p105)” instead of “NF-κB(105)”.
7. Line 177 Figure 1 or other possible figures: Did the authors detect EGFR protein expression (western blot) in PC9 and PC9/GR after treatment with Gefitinib? If yes, provide data. EGFR and p-EGFR expressions should be included.
8. Line 178: I’d suggest the authors use an additional panel to present IL-6 mRNA expression. For example, Figure 1D, or whatever the authors prefer.
9. Figure 1: Please add arrows to the target band if there are multiple bands shown in the image. The concentration unit “uM” should be “µM”. The statistical method should be listed in the figure legend.
10. Line 201 Figure 2: Please see the concerns above.
11. Line 220 Figure 3: Please see the concerns above.
12. Line 244 Figure 4: Please add scale bars to Figure 4A.
13. Line 246: Please keep consistent with or without space before and after equal sign throughout the manuscript.
14. Lines 248-249: Please add a reference to “Initially, patients with EGFR-mutated NSCLC show a good response to EGFR-TKI therapy; however, the tumors inevitably acquire drug resistance.”
15. Lines 326-329: Why and what is the meaning of the following sentences in the conclusions section “Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.”

Reviewer 2 Report

Reviewer comments:

Comments to the Author

This manuscript aimed to determine the expression of NF-κB, AICDA, Akt, IL-6, Jak2, and Stat3 by EGFR-TKI-resistant lung adenocarcinoma (LAC) and found that NF-κB and AICDA are major players in acquired resistance of lung cancer to TKIs. This is a well conducted study that raises new concepts regarding cosuppression of NF-κB and AICDA may be a promising strategy to overcome EGFR-TKI resistance in LACs.

The experimental designing is impressive, and the manuscript is for the most part well written with substantial evidence of confirmatory and supplementary data. The discussion is also well goes with the results and postulated according to the evidence provided. The references are appropriate and timely.

 

Minor criticisms

• Please provide the information about dose selection for Gefitinib and NFkB inhibitor involved in these studies.
• Information about number of samples used to conduct each experiment is not provided In the figure legend of figure1, 2 and 3. Authors are advised to provide N values for each experiment in the figure legends.
• Please undergo a thorough check of the manuscript for typographical and grammatical errors.

 

 

 

 

 

 

Reviewer 3 Report

Reviewer comments:

Comments to the Author

The manuscript entitled “Cosuppression of NFκB and AICDA overcomes acquired EGFR-TKI resistance in non-small cell lung cancer” by Min- Kyung et al., provides an interesting insight to possible treatment with an EGFR-TKI plus cosuppression of NF-κB and AICDA may be a promising strategy to overcome EGFR-TKI resistance in LACs.

This study provides novel evidence to the mechanistic basis for the treatment with an EGFR-TKI plus cosuppression of NF-κB and AICDA could be a promising strategy to overcome EGFR-TKI resistance in LACs.

Further details required in the methods or supplementary methods:

• Whether authors have analyzed the histological data on other proteins such as RELB, AICDA, IL-6, Jak2, Stat3, Akt, and cytidine deaminase. Need to present data on these markers and discuss it in the result section.
• Whether the dose for Gefitinib and other inhibitor was selected based on the viability of the cells. Please include data and condition of the cells.
• Authors should provide the sample number or number of replicates for Western blotting data and mention it in all the figure legends. How reproducible are the Western blots?
• A thorough check for typographical and grammatical errors should be performed throughout the manuscript.