PONE-D-20-04249

PHRF1 promotes migration and invasion by modulating ZEB1 expression

PLOS ONE

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Additional Editor Comments (if provided):

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

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 ms by Lee and colleagues entitled “PHRF1 promotes migration and invasion by modulating ZEB1 expression” describes a novel mechanism of ZEB1 regulation by PHRF1, a protein with an important role in APL tumorigenesis, contributing to EMT process in lung cancer cells. The authors have checked the levels of PHRF1 in different tumors using a publicly accessible database and have associated them with overall and progression free survival. Furthermore, authors have studied the effect of PHRF1 silencing and PHRF1 overexpression on cell migration and invasion, as well as on the levels of EMT markers, such as E-cadherin and N-cadherin suggesting an important role of this protein in the lung cells malignant transformation. Authors also confirmed that ZEB1 is responsible for PHRF1-mediated migration and invasion analyzing the silencing of ZEB1 in PHRF1- overexpressing lung cells. Finally, direct PHRF1 binding adjacent to the TSS region of ZEB1 was confirmed in ChIP-PCR. In summary, data presented by Lee and colleagues suggest that PHRF1 could modulate the expression of ZEB1 promoting migration and invasion in cancer lung cells.

Overall, the data in this ms are novel and important, nevertheless I see several defficiencies that should be addressed:

Major comments:

1. Abstract and Introduction: I suggest to rewrite all and I recommend a revision by native speaker. There are some nonsense sentences as in line 25 "The C-terminal Set2 Rpb1 Interacting (SRI) domain…”, I really do not understand what you are trying to mean. Probably, it is incorrectly written, so please, rewrite all trying to make sense of the text and with a clear and concise message.

I feel the same in the Introduction section where there are a huge amount of irrelevant information and where it is impossible to get any conclusion (lines 55 to 66).

2. The sequences of primers you listed in S1 table are wrong; they have no targets or they match with predicted targets that are not the genes that supposed to be. Please, recheck all the sequences.

3. Have the authors blocked proliferation when performing migration assays? In this sense, I think 0’5% of FBS is a better way to avoid proliferation in this kind of experiments.

4. In results the authors resume the association of PHRF1 expression with overall and progression free survival in a number human tumors that are so far in publicly database. Regarding this point it would be interesting to do an analysis of expression in TCGA datasets using tools like cbioportal, firebrowse, etc. I also suggest, using the TCGA database and the available online tools, to do perform an analysis of whether the expression of this protein correlates with the clinical attributes of the tumor. This point could improve the study and could support the data presented in the manuscript.

5. In Western blots reported in Figure 1E, densitometry with statistical analysis must be added. Regarding this point, it is remarkable that control cells in silencing experiments appear to have reasonable PHRF1 protein expression in comparison to control cells in overexpressing experiments. Do the authors have an explanation for this differences in the expression? Finally, I missed the N-cadherin protein expression in the silencing experiments. Did you analyze N-cadherin expression in the PHRF1 silenced cells?

6. In western blots reported in Figures 3A and 3B please add densitometry with statistical analysis.

7.Fig 4A. Densitometry with statistical analysis must be added.

8. ChIP-PCR (Fig. 4E): an important negative control in ChIP experiments is a distant region in cis to the putative binding site (e.g. a kb away from the promoter). Could this be included?

10. You cite a paper of Wang et al. (2016) in the Introduction demonstrating a tumor suppressor role of PHRF1 in lung cancer, however, I am surprised that these results are not discussed in the Discussion section. How do you explain these controversial data?

Minor comments:

1. Use capital letters for human genes: ZEB2 in lines: 250, 252, 253, 254, 291, 303 and 323.

2. All human genes have to be in italics.

3. In line 269: “PHRF1 and ZEB1 were induced in control cells under hypoxia; nonetheless, the amount of ZEB1 was unable to increase in PHRF1 depleted cells under low O2 condition (S5A Fig).”; text does not correspond with the figure, and the figure S5A is missing.

Reviewer #2: The authors of this paper carried out a very interesting investigation of the implications behind PHRF1 potentially acting as a promoter of migration and invasion via ZEB1 expression. They used a variety of well thought-out experiments to show this, including overexpressing and knocking-down expression of PHRF1 in lung cancer cells and seeing their effects on Zeb1 and migration and invasion, among other factors implying tumorigenicity. They also carry out ChIP in order to see binding of PHRF1 to facilitate RNA Pol 2 transcription/elongation to further strengthen their claim that PHRF1 may be capable of regulating the trx of genes like Zeb1.

While the study is a very fascinating one, please consider the following suggestions.

1) There are several English expression errors throughout the paper. Please revise and make sure to correct.

2) While this paper is full of different molecules and the authors generally do a good job at defining each molecule before using the abbreviation, they don’t do so for PHRF1. Please do so. And also NBS1. What does this stand for? Those were 2 molecules that I noticed but please go over to make sure haven’t missed any others.

3) For both the Abstract and the Introduction, please make a better connection between what is known about PHRF1 and your interests for studying it as a promoter of tumor invasion/migration/progression. Ettahar A et al 2013 “Identification of PHRF1 as a tumor suppressor that promotes the TGFB cytostatic program through selective release…” seems to suggest PHRF1 may work as the exact opposite of what this paper is seeing. Please explain this conflicting observation. What is known about PHRF1? Is work the first to suggest PHRF1 may actually not be a tumor suppressor? What have others published?

4) For Figure 1E, this piece of data especially for E-cadherin is ok but not the most convincing. Have you tried looking at other EMT markers perhaps? To show EMT is occurring?

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Reviewer #1: No

Reviewer #2: No

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PHRF1 promotes migration and invasion by modulating ZEB1 expression

PONE-D-20-04249R1

Dear Dr. Mau-Sun Chang,

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.

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Olorunseun Ogunwobi, MD, PhD

Academic Editor

PLOS ONE

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

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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

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

Reviewer #1: 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

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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

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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: (No Response)

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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