PONE-D-20-37563

Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant hepatocellular carcinoma cells

PLOS ONE

Dear Dr. Morine,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Olorunseun Ogunwobi, MD, PhD

Academic Editor

PLOS ONE

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1. Thank you for stating in your Funding Statement:

This study was partly supported by Research Program on Hepatitis from the Japan Agency for Medical Research and Development (AMED) Grant Numbers JP19fk0210048 and JP20fk0210048. And co-authors (Shimada M) received a research grants from Grant-in-Aid for Scientific Research (B) (KAKENHI), Project code: 18K02871, during the conduct of the study.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have shown that Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant hepatocellular carcinoma cells. The work is interesting, however there are several drawbacks.

Major concern:

1. Introduction portion is not well written and needs to be improved.

2. You have given references related to KEAP1/ NRF2 pathway and the role of CSCs in cancer recurrence and chemotherapy resistance in the result section. These should be mentioned in the introduction section.

2. Figure quality is poor. I will suggest you to provide better figures for wound healing assays (Figure 2 and 6).

3. All the conclusions are based only on RT-PCR data. You should provide western blot data for further validation.

Minor mistake:

1. What do you mean by “ideal” confluency in the wound healing assay?

2. The sequence of siRNA and primers are missing.

3. Mention the amount of total RNA used in cDNA synthesis.

4. Please provide the PCR protocol in details.

Reviewer #2: In this manuscript, Gao et al. demonstrate that cancer stemness, migration/invasion and expression of ABC transporter genes are upregulated in the sorafenib-resistant hepatocellular carcinoma cells (HCC) they generated. Knockdown of Nrf2 reduced cancer stemness, migration/invasion and expression of ABC transporter genes in the sorafenib-resistant HCC cells. The authors propose that targeting Nrf2 signaling pathway has therapeutic potential in sorafenib-resistant HCC. This is an interesting work and it has been shown overexpression of Nrf2 in HCCs and Nrf2 regulates oxidative stress response and drives tumorigenesis. However, some additional experiments and modifications/data interpretation to the manuscript should be addressed to strengthen the discovery.

Fig.1A Is the difference of cell survival rate between Huh7 and Huh7-res cells statistically significant?

Fig.1B Methodological detail of Fig. 1B is missing. Furthermore, no precise measurement of the difference of cell volume or cytoplasm between Huh7 and Huh7-res cells and statistical analysis. In addition, please indicate vacuole structures.

Fig. 2A&B Fig2C showed higher proliferation of Huh7-res cells vs. Huh7 cell in the presence of sorafenib in a 3-week assay. Are there any proliferation data in short time period, with and without sorafenib? Does the proliferation difference affect the results of migration and wound healing assay in 24h?

Fig.3 What are the mRNA levels of EPCAM, Nanog, CD44 in tumorsphere assay, a three-dimensional culture setting between Huh-7 and Huh-res cells? In addition, this experiment lacks methodological detail for other researcher to repeat the experiment, what is the PCR primer set for EPCAM, Nanog and CD44. Anther question is what are the mRNA levels of EPCAM, Nanog, CD44 in the presence or absence of sorafenib in Huh7-res cells?

Fig. 4. What are the protein levels of Nrf2, HO-1, ABCC1 ABCG2 and ABCB1 and ABCA6? Western blot showing protein levels of these proteins will strengthen this part of the paper.

Fig. 5 A non-targeting siRNA control is not included, but that needs to demonstrate the reduction of Nrf2, CD44, Nanog was not because of transfection itself.

Fig.6 Similar to the point above, how cell proliferation was affected by knockdown Nrf2? Again, is the invasion or migration measured here because of cell proliferation?

Ultimately, all assays were performed in a single cell line, Huh7. Minimally these experiments should be repeated in at least one additional HCC cell line and its sorafenib-resistant cell line to establish how general this phenomenon is.

Overall, the publication of this manuscript at this time is a bit premature.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-37563R1

Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant hepatocellular carcinoma cells

PLOS ONE

Dear Dr. Morine,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please ensure that all comments and concerns included in the reports from both reviewers are fully addressed.

==============================

Please submit your revised manuscript by Aug 16 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Olorunseun Ogunwobi, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript has been improved significantly. However, you have to take care of a few things to improve it further.

1. The figures of wound healing/ migration assays are still not clear.

2. The HO-1 western blot image is not good.

3. The fonts used in the figures are hazy. Please try to make it clearly visible. The supplementary figures' fonts are better than the fonts used in the main figures of the manuscript.

4. Please use the same font type throughout your manuscript.

5. Some typos are there in the manuscript, for eg,

page 58, line 3: “Nrf2 helps maintain the oxidative stress balance”

page 63: “asaay”

page 64: “Nuceis”

Reviewer #2: The authors have clearly responded to my questions and made the necessary changes. I would like to recommend the revised version of the manuscript for publication. Minor points/comments regarding the figures:

Fig. 1a In the text it states: The IC50 of Huh7 was 10.68μM, IC50 of sorafenib resistant Huh7 was 16.48μM; however, in the figure the x axis label for Sorafenib log concentration is nM.

Fig. 2a right panel missing "-" in Sorafenib treatment line.

Fig. 6b: the quality of the figure needs improvement.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Nrf2 signaling promotes cancer stemness, migration, and expression of ABC transporter genes in sorafenib-resistant hepatocellular carcinoma cells

PONE-D-20-37563R2

Dear Dr. Morine,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Olorunseun Ogunwobi, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed al the comments and the manuscript has been improved significantly. I will suggest you to provide the sequences of siRNAs used in this study.

Reviewer #2: The authors have made the changes that I've recommended. The revised version of the manuscript is suitable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No