PONE-D-20-03351

PD173074 blocks G1/S transition via CUL3-mediated ubiquitin protease in HepG2 and Hep3B cells

PLOS ONE

Dear Dr. Chuchu Qiao,

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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ACADEMIC EDITOR: This article is aimed to study the role PD173074, a FGFR inhibitor, in HCC. This article is interesting and contained novelty. However, there are some major concerns for this article. Those points should be clarified.   

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We would appreciate receiving your revised manuscript by Apr 24 2020 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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Kind regards,

Yu-Jia Chang

Academic Editor

PLOS ONE

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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: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: 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: PD173074 blocks G1/S transition via CUL3-mediated ubiquitin protease in HepG2 and Hep3B cells

Limitations:

(a) This study lacks clinical relevance to assess the significance of molecules in the FGFR4 mediated ERK/CUL3/cyclin E signaling pathways.

(b) PD173074 is a pan-FGFR inhibitor, not a specific FGFR inhibitor. It has been reported that BLU9931, a potent and irreversible small-molecule inhibitor of FGFR4. The authors do not compare the cytotoxicity efficiency of PD173074 with any other well-known FGFR inhibitor. In this study, these HCC cell lines only express FGFR4. It’s unknown whether specific FGFR4-mediated ERK/CUL3/cyclin E axis that leads to cell proliferation.

Strengths:

(a) In this study, the authors used PD173074, an inhibitor of FGFRs, to explore the underlying mechanism of FGFRs in HCC cells, and the data indicated that FGFR4 may be involved in the proliferation of HCC via ERK/CUL3/cyclin E axis.

Major comments:

1. Previous reports showed that FGFR1 and FGFR2 were shown to be elevated in the majority of HCC cells. Aberrant FGF19 signaling through FGFR4 has been identified as an oncogenic driver for a subset of patients with HCC. FGF19 and its receptor FGFR4 have been shown to be involved in EMT in HCC cells through modulating the GSK3β/β-catenin signaling cascade. The authors can provide the clinical outcome association of specific FGFR expression (FGFR4) in HCC via public microarray dataset analysis. Otherwise, the rationale is weak if no data showed poor outcome of FGFR4 overexpression in HCC progression. For example, it needs to clarify whether the expression levels of FGFR4 are correlated with HCC progression (eg. tumor size, metastasis, and angiogenesis etc.). Additionally, in this study it also can’t reveal the clinical correlation of FGFR4 with downstream targets including cyclin E, CUL3 or miR-141 expression in HCC progression.

2. In the Figure 1, the author examined the effect of PD173074 on the viability of HepG2, Hep3B and normal liver cell line HL7702 cells. Actually, high concentration of (>20 uM) PD173074 can induce most cell death in these cells. 0.5-10 uM PD173074 can lead to significant HCC cell death. However, it is unknown the cytotoxicity efficiency of PD173074 compared with another FGFR inhibitor. Also, the action of PD173074 has not been characterized in this study.

3. Previous study showed that FGFR1 and FGFR2 expression can be examined in HCC SK-Hep-1 and SNU449 cells. (Mol Cancer Ther. 2015;14(11):2613-2622. PMID: 26351320). In the Figure S1A, there is no detectable level of FGFR1 in HepG2, Hep3B and HL7702 cells. However, FGFR4 is overexpressed in HepG2 and Hep3B cells compared with normal control. It’s unknown whether the specific FGFR4 may lead to liver cancer cell proliferation via ERK/CUL3/cyclin E axis dependent on different cell context.

4. The study demonstrates that FGFR4 is involved in the proliferation of HCC via ERK/CUL3/cyclin E axis. The authors would like to provide a potential theoretical basis for treatment by targeting FGFR4 in HCC. PD173074 seems to be a pan-FGFR inhibitor and whether another pan-inhibitor (eg. AZD4547) or selective inhibitors (eg. BLU9931) have similar effect in ERK/CUL3/cyclin E axis. Actually, a number of FGFR inhibitors are currently in clinical trials to treat cancers with FGFR. What the efficacy of potential FGFR inhibitors has been examined for HCC therapy in recent clinical trials?

Minor:

In the page 17, line 229-230, misspell of “detectible”.

In the Figure 2B, the concentration of PD173074 should be noted in the experiment.

Reviewer #2: This manuscript investigates whether PD173074, an FGFR inhibitor can be used as an HCC anti-cancer agent. The authors used HCC cell lines HepG2, Hep3B and one normal liver cells demonstrated PD173074 inhibiting FRS2α, ERK and downstream signals, as well as cyclin E regulation. This is a very interesting and well-design study. Some minor typos or mistakes are given to improve the quality of this study.

1: This manuscript is strongly suggested to do English professional editing. Several grammatical mistakes are consistently found through the context.

For example:

little change was found in HL7702 cells-236

The meanwhile-273

2: Typos: Detectible-229, 230

3: What is UPS: No definition is shown whole the context.-267, 268

4: Since the authors used p-FRS2α in this study, the phosph-FRS2α should be clearly identified in this manuscript, for example: line 233, 235, 239….. And the total FRS2α detection should be also included in figure 2.

Reviewer #3: This paper by a group of researchers from China sheds light onto the potential therapeutic strategy of FGFR inhibitor, PD173074, in hepatoma. Recently, alterations of FGFR have been reported to be important for progression and development of several cancers, including lung cancer and breast cancer. Authors tried to figure out the puzzle of the molecular signaling pathway regulated by FGFR/PD173074. However, there’s still lots of missing pieces in this entire picture.

The vast part of the data in this manuscript is to understand the therapeutic effect of PD173074 in FGFR-overexpressed hepatoma cell lines, but no in vivo-related data to prove the crucial role of FGFR in hepatoma. Authors may provide some evidence to reveal the effects of PD173074 in in vivo study (animal model or clinical evidence).

PD173074 is known to be a selective inhibitor of FGFR1 (doi: 10.1038/bjc.2013.550). However, there’s undetectable endogenous FGFR1 in HepG2 and Hep3B hepatoma cell lines. And authors did not provide the data of FGFR2 and 3 in the cell lines. How could we exclude the compensation effects from FGFR2 and 2 in this study?

The threaraprutic window is unsatisfied in low concentration of PD173074 (1 µM) in Figure 1. Previous studies \\used to use low concentration to st\\dy the effects of PD173074 in anti-cancer experiments. Nguyen et al., used 15 nM PD for 1h (doi: 10.1038/bjc.2013.550), and Pardo et al., used 10nM PD for 1h (doi: 10.1158/0008-5472.CAN-09-1576). Could authors discuss the differences between this?

Signal transduction is very fast and the phospho-protein will go back to the basal level after stimulation, unless the constitutive activation mutantion is present.

The response time of signal transduction protein and cell cycle regulator to extracellular ligand stimulation are totally different. Authors did not clearly point out the suitable time point to observe the activation status of signal transduction protein and cell cycle regulator, respectively.

Minor point:

The use of the English is not always appropriate, and the manascript would be benefit from some careful revision with respect to syntax, grammar and typos.

Could author provide the proliferation rate of the three cell lines used in this study? According to Figure 1, the population doubling time of hepatoma cell lines and normal liver cell line seems the same?

Please label the exact time point and PD173074 concentration in different concentration experiment and time course experiment, respectively.

There are some typos in the article, please check it.

1. In P. 29, the year of reference 5, please check it

2. Is the cell line named Hep3B or Hep3b? Please check it.

3. In row 217, there are two “blocks”.

4. In Figure 1B, the concentration in colony formation are 0.5, 1 and 10 µM or 1, 10 and 50 µM?

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Reviewer #1: No

Reviewer #2: Yes: Chia-Hwa Lee

Reviewer #3: 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 to be viewed.]

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PONE-D-20-03351R1

PD173074 blocks G1/S transition via CUL3-mediated ubiquitin protease in HepG2 and Hep3B cells

PLOS ONE

Dear Dr. Qiao_Chuchu,

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.

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

ACADEMIC EDITOR: The authors have been improved the quality of the manuscript. However, there are some minor concerns for the revised manuscript.

We would appreciate receiving your revised manuscript by Jun 25 2020 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,

Yu-Jia Chang

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The authors have been improved the manuscript. However, there are some minor concerns. Please add scale bars into each figure panel and size markers to each western blot. I also strongly encourage you to upload your original data to an appropriate figure/data repository such as Mendeley Data or Dryad, as access to original data can increase reader confidence in the findings.

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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

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

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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

Reviewer #3: (No Response)

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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

Reviewer #2: Yes

Reviewer #3: (No Response)

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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: Although there is no clinical evidence in this study, it revealed that FGFR4 may affect ERK/CUL3/cyclin E signaling pathway in HCC cells through basic study.

Reviewer #2: (No Response)

Reviewer #3: Please add scale bars into each figure panel and size markers to each western blot. I also strongly encourage you to upload your original data to an appropriate figure/data repository such as Mendeley Data or Dryad, as access to original data can increase reader confidence in the findings.

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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.

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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: Yes: Sheng-Ming Wu

Reviewer #2: Yes: Chia-Hwa Lee

Reviewer #3: 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 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.

PD173074 blocks G1/S transition via CUL3-mediated ubiquitin protease in HepG2 and Hep3B cells

PONE-D-20-03351R2

Dear Dr. Qiao_Chuchu,

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.

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Yu-Jia Chang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: