PONE-D-19-20240

IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

PLOS ONE

Dear Dr. Lee,

Thank you for submitting your manuscript to PLOS ONE.  I have received to date one completed review and could not get a second one. I have personally evaluated your article and I totally agree with the comments raised by the reviewer. Based on these recommendations, and 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.

Specifically, the reviewer has found that your study contains interesting findings but also underlined significant weaknesses in the methodology. You should precisely answer to his 10 distinct comments. A particular attention will be paid to the concerns related to methodological and statistical issues as well as interpretation of the data. I also think that the use of a loss of-function approach with anti-miR would be instrumental to assess the function of the endogenous miRNA on TMZ sensitivity as well as on its target. Regarding the last point raised by the reviewer (glioma stem cells), while I think the use of patient-derived glioma stem cells would strengthen the scope of the study, it will not represent an essential criterion for the final decision.  Finally, I have also personally raised one additional point: while the authors state that all data are fully available, they do not provide a link for the microarray dataset. The whole miRNA microarray dataset should be deposited to a public database such as Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). 

We would appreciate receiving your revised manuscript by Oct 06 2019 11:59PM. When you are 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Bernard Mari, Ph.D

Academic Editor

PLOS ONE

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Reviewer #1: Partly

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

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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5. Review Comments to the Author

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Reviewer #1: In this article the authors have identified in GBM cell lines, a novel IGF1 dependent molecular mechanism in where miR-513a-5p represses NEDD4L leading to activation of the WNT/beta-Catenin pathway. This beta-Catenin activation would confer higher resistance to TMZ treatment. The study is interesting but needs more development before to jump too fast at the conclusion. See specific comments below.

Comments to the authors :

In the figure 1, the authors need to clearly demonstrate beta-catenin nuclear translocation upon IGF1 treatment. This could be done by immunofluorescence or by cell fractionation. Also showing beta-Catenin nuclear localisation is not enough to conclude to an increase of its activation upon IGF1. The authors need to assess its transcriptional activity upon IGF1 treatment by using a reporter gene containing TCF/LEF binding sites (top flash reporter gene).

The cell proliferation has been determine by MTT assay which reveals only 13% increase of cell proliferation upon IGF1. This is an indirect method to measure cell proliferation which need to be confirmed by counting viable cells following IGF1 treatment.

Figure 1 shows an increase of cyclin D1 expression upon IGF1 treatment. Although cyclin D1 is a well known target of beta-Catenin many other factors may stimulate this gene upon growth factor stimulation. The authors have to show that this increase is beta-Catenin dependent by performing its functional invalidation using specific siRNA.

Similar functional invalidation need to be conducted to assess whether or not the 13% increase of proliferation is due to WNT/beta-Catenin activation.

Cell sensitivity to TMZ mediated death has been carried out by using MTT assay. A direct method to measure cell death will be much more appreciated.

Beta-Catenin invalidation need to be performed to confirm its contribution in TMZ resistance. This will also confirm the results obtained with GSK3-beta inhibition which is not functionally obvious in the case of M059K cells. The quantification of DNA damages would be also informative in this context.

The authors show increased miR-513a-5p upon IGF1 stimulation. It would be interesting to assess the effect of miR-513a-5p invalidation (anti-miR) on GBM cell sensitivity to TMZ following IGF1 treatment.

The results clearly indicate that NEDD4L 3’UTR can be bound by miR-513a-5p. MiR-513a-5p overexpression induces the decrease of NEDD4L protein. These results are convincing but based on gene overexpression. In order to confirm the physiological relevance of these results, the authors need to perform miR-513a-5p invalidation (upon IGF1 treatment) and evaluate NEDD4L expression.

The consequences of miR-513a-5p invalidation on beta-Catenin nuclear localization need also to be assessed to confirm the relevance of this miR for activation of this pathway.

To validate their study, the authors have to reproduce at least part of their data in patient-derived glioma stem cells which are definitely much more relevant to study GBM physiology than GBM cell lines.

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Reviewer #1: Yes: Thierry Virolle

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PONE-D-19-20240R1

IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

PLOS ONE

Dear Dr. Lee,

Thank you for resubmitting your manuscript to PLOS ONE. While you have adequately addressed most of the queries in the review and that the revised manuscript is significantly improved from its original submission, one issue still needs to be addressed before full acceptance of the paper.

Specifically:

As mentioned during the first review, the authors should provide a link for the microarray dataset. The whole miRNA microarray dataset should be deposited to a public database such as Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) and the GSE reference should be included in the material and method section.

We would appreciate receiving your revised manuscript by Dec 27 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Bernard Mari, Ph.D

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway

PONE-D-19-20240R2

Dear Dr. Lee,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Bernard Mari, Ph.D

Academic Editor

PLOS ONE

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