GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathway

Xiang Guo; Zongliang Guo; Peirong Bai; Congfang Guo; Xuewei Liu; Kaiyi Zhu; Xiaoyan Li; Yiyan Zhao

doi:10.1371/journal.pone.0302061

Peer Review History

Original SubmissionSeptember 14, 2023
2 Nov 2023 Decision Letter - Suzie Chen, Editor PONE-D-23-27240GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathwayPLOS ONE Dear Dr. Zhao, 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 submit your revised manuscript by Dec 17 2023 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: https://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, Suzie Chen Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: - https://www.nature.com/articles/s41419-018-0299-1#citeas In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. 3. Thank you for stating the following financial disclosure: "GX: the Fundamental Research Program of Shanxi Province (202203021222348), National Natural Science Foundation of China (8230089), and Scientific research start-up fund project for talent introduction of Shanxi Bethune Hospital (2023RC22). Xiang Guo designed the experiments, performed most of the experiments and wrote the paper." Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 6. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 7. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Additional Editor Comments: Three reviewers have carefully review your manuscript and suggested major revisions. Please read each of their comments carefully and answer each concern point by point, if necessary please also provide supporting data. Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact. For Lab, Study and Registered Report Protocols: These article types are not expected to include results but may include pilot data. [Note: HTML markup is below. Please do not edit.] 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: No Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know Reviewer #3: Yes ****** 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 ****** 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: No Reviewer #3: Yes ****** 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: In this current article titled "GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathway", authors reported the tumor suppressing properties of GPR168 in mouse melanoma. The paper contains its own merit considering the findings of a noble targets of melanoma however, it requires MAJOR REVISION before considering for publications. (1) Its unrelated to show TCGA human data set while validate with murine cell lines. Author should consider incorporation of at least one human melanoma cell lines. (2) The data extracted from GEPIA and cBioportal even unrealistically matched with current findings as TCGA melanoma cohort published in 2015 by pancancer atlas considered both primary and metastatic tumor for their study. This cohort doesn't purely represent the story related with malignant melanoma. (3) What's the rationale of overexpression total AKT expression in pCDH-GPR168 cells? (4) After blocking the function of GPR168, p-AKT expression didn't change and cell viability didn't alter compared with empty vector. How this target can be trusted to became a control factor of tumorigenesis? (5) The figures look blurred. Reviewer #2: Although the bioinformatics analysis shows a decreased expression of GPR168 across different human tumors including melanoma, the paper falls short of showing a causality of low expressed GPR168 on melanoma.The entire paper relied on one mouse melanoma cell line (B16-F10) with or without ectopic expression of GPR168. There are inconsistencies in the results even with this cell line. The authors mistakenly considered injecting mouse melanoma cell line F10 into nude mice an allogeneic model. Reviewer #3: The manuscript “GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathway” by Guo et al is very interesting as there are only a few tumor suppressors. Please see below my specific comments. Specific Comments: 1. Expression of GPR in B10-F10 should be shown by immunoblotting. Otherwise, we assume that it is there without any proof. 2. CDK4 is down but no change in cyclin D1. Also, how then there is a decrease in Ki67? Minor Comments: Figures are of poor quality especially IHC. Effort must be made to produce high quality figures. ****** 6. 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 Reviewer #3: Yes: Dayanidhi Raman ******** [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. https://doi.org/10.1371/journal.pone.0302061.r001
Revision 1
11 Jan 2024 Author Response Responses to Reviewer 1 In this current article titled "GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathway", authors reported the tumor suppressing properties of GPR168 in mouse melanoma. The paper contains its own merit considering the findings of a noble targets of melanoma however, it requires MAJOR REVISION before considering for publications. Response: Thanks for reviewer’s comments. We have made MAJOR REVISION according to your suggestions. 1. Its unrelated to show TCGA human data set while validate with murine cell lines. Author should consider incorporation of at least one human melanoma cell lines. Response: We applied Cross-Value Association Analysis (CVAA) to large-scale pan-cancer transcriptome data generated by The Cancer Genome Atlas (TCGA) [1] and found that GPR168 is one of Differentially Expressed Genes (DEGs). Subsequently, we found that only one paper reported that GPR168 (MrgprF) acts as a tumor suppressor in cutaneous melanoma by restraining PI3K/Akt signaling pathway, and this result was confirmed by four human melanoma cells [2]. In order to further verify whether GPR168 involves in the PI3K/Akt signaling pathway, mouse melanoma B16-F10 cells were used to perform cell experiments in vitro and xenograph tumor models in vivo. 2. The data extracted from GEPIA and cBioportal even unrealistically matched with current findings as TCGA melanoma cohort published in 2015 by pancancer atlas considered both primary and metastatic tumor for their study. This cohort doesn't purely represent the story related with malignant melanoma. Response: Thanks for reviewer’s professional comments. As you said, this cohort doesn't purely represent the story related with malignant melanoma. However, we want to collect as much public data as possible to make a preliminary analysis of whether GPR168 is related to melanoma. Therefore, we used as many melanoma data from multiple databases as possible to make a preliminary statistical analysis of the correlation, making it more statistical and scientific to show that GPR168 is associated with melanoma. However, this result could not prove that GPR168 is directly related to melanoma, so we performed a series of follow-up experiments in vitro and in vivo (Fig 2-4). 3. What's the rationale of overexpression total AKT expression in pCDH-GPR168 cells? Response: Thanks for reviewer’s question, which allows us to discover new problems. We repeated the western blotting experiments and found that the expression of AKT in pCDH-Vec and pCDH-GPR168 had no significant difference (Fig 3F). Fig 3. Overexpression of GPR168 inhibited xenograft tumors growth in vivo. (A) The sizes of allogenic tumors at day 30 were smaller by pCDH-GPR168, compared to those by pCDH-Vec. (B) GPR168 overexpression significantly reduced MM tumor growth in nude mice by tumor volume examination from 0 to 30 day. (C) Overexpression of GPR168 significantly suppressed the weights of allogeneic tumors. (D, E) the positive cells of Ki67 in allogenic tumor tissues. Scale bar is 100 μm, (E) Quantification for (D). (F) Akt, p-Akt, GSK-3β, p- GSK-3β, β-catenin, Myc, CyclinD1, CDK4 protein expression in pCDH-GPR168 group and pCDH-Vec group (Western blotting). (G) β-catenin, Myc, CyclinD1, CDK4 mRNA expression in pCDH-GPR168 group and pCDH-Vec group cells (RT-qPCR). (H) A signaling pathway model for GPR168 function in B16-F10 cell line. β-actin as normalized protein. P < 0.05, P < 0.01, *P < 0.001, t-test. 4. After blocking the function of GPR168, p-AKT expression didn't change and cell viability didn't alter compared with empty vector. How this target can be trusted to became a control factor of tumorigenesis? Response: The overexpression of GPR168 could inhibit the proliferation and migration of melanoma, as well as the formation of xenograft tumors (Fig 2). After a series of functional experiments, it was found that GPR168 could function through the AKT signaling pathway (Fig 3). We preliminarily concluded that GPR168 is a tumor suppressor (melanoma) gene. Subsequently, we performed rescue experiment, and found that Anti-GPR168 pAbs could block the function of pCDH-GPR168 plasmid in B16-F10 tumor cells (Fig 4), which further confirmed that GPR168 could inhibit the tumorigenesis of melanoma through AKT signaling pathway. Taken together, we conclude that GPR168 could be a control factor of tumorigenesis (melanoma). 5. The figures look blurred. Response: Thanks for reviewer’s comments. We repeated Western blotting experiment and re-made the Fig 1D, 2B, 3F and 4C. Responses to Reviewer 2 Although the bioinformatics analysis shows a decreased expression of GPR168 across different human tumors including melanoma, the paper falls short of showing a causality of low expressed GPR168 on melanoma. The entire paper relied on one mouse melanoma cell line (B16-F10) with or without ectopic expression of GPR168. There are inconsistencies in the results even with this cell line. The authors mistakenly considered injecting mouse melanoma cell line F10 into nude mice an allogeneic model. Response: Thanks for reviewer’s comments. In this paper, we found that the expression of GPR168 might be correlated with the tumorigenesis of melanoma (Fig 1). Subsequently, we found that one paper reported that GPR168 (MrgprF) acts as a tumor suppressor in cutaneous melanoma by restraining PI3K/Akt signaling pathway, and this result was confirmed by four human melanoma cells [2]. In order to further explore the function of GPR168 in melanoma cells, cell experiments in vitro (Fig 2) and xenograph tumor models in vivo (Fig 3) were performed and the results showed the overexpression of GPR168 could inhibit the tumorigenesis of melanoma and GPR168 involved in the PI3K/Akt signaling pathway. Furthermore, we confirmed this conclusion by rescue experiment (Fig 4). Responses to Reviewer 3 The manuscript “GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathway” by Guo et al is very interesting as there are only a few tumor suppressors. Please see below my specific comments. Specific Comments: 1. Expression of GPR in B10-F10 should be shown by immunoblotting. Otherwise, we assume that it is there without any proof. Response: Thanks for reviewer’s comments. We have performed Western blotting as reviewer’s suggestion. The western blotting results showed that the B16-F10 cell line with stable GPR168 expression was successfully constructed (Fig 2B). Fig 2. Overexpression of GPR168 inhibited B16-F10 cell proliferation and migration. (A, B) The efficiency of pCDH-GPR168 (GPR168 overexpression) was verified by RT-qPCR and western botting, compared to scramble pCDH-Vec. (C, D) GPR168 overexpression inhibited cell proliferation in B16-F10 cells by Ki67 Immunofluorescence assay. Scale bar is 100 μm. (E, F) Result of CCK-8 assay (E) and Growth Curve (F) showed that overexpression of GPR168 significantly inhibited B16-F10 cell proliferation. (G, H) overexpression of GPR168 decreased the migration ability of B16-F10 by transwell assay at OD570. Scale bar is 100 μm. (I, J) Wound healing assay showed that overexpression of GPR168 inhibited the migration of B16-F10 cells. Scale bar is 100 μm. P < 0.05, P < 0.01, P < 0.001, t-test. 2. CDK4 is down but no change in cyclin D1. Also, how then there is a decrease in Ki67? Response: Thanks for reviewer’s question, which allows us to discover new problems. We repeated the western blotting experiments and found that both of CDK4 and cyclin D1 protein were decreased (Fig 3F). Fig 3. Overexpression of GPR168 inhibited xenograft tumors growth in vivo. (A) The sizes of allogenic tumors at day 30 were smaller by pCDH-GPR168, compared to those by pCDH-Vec. (B) GPR168 overexpression significantly reduced MM tumor growth in nude mice by tumor volume examination from 0 to 30 day. (C) Overexpression of GPR168 significantly suppressed the weights of allogeneic tumors. (D, E) the positive cells of Ki67 in allogenic tumor tissues. Scale bar is 100 μm, (E) Quantification for (D). (F) Akt, p-Akt, GSK-3β, p- GSK-3β, β-catenin, Myc, CyclinD1, CDK4 protein expression in pCDH-GPR168 group and pCDH-Vec group (Western blotting). (G) β-catenin, Myc, CyclinD1, CDK4 mRNA expression in pCDH-GPR168 group and pCDH-Vec group cells (RT-PCR). (H) A signaling pathway model for GPR168 function in B16-F10 cell line. β-actin as normalized protein. P < 0.05, P < 0.01, P < 0.001, t-test. 3. Minor Comments: Figures are of poor quality especially IHC. Effort must be made to produce high quality figures. Response: Sorry for poor quality of figures. We have made revise in Fig 3D. References: [1] Li QG, He YH, Wu H, Yang CP, Pu SY, Fan SQ, et al. A normalization-free and nonparametric method sharpens largescale transcriptome analysis and reveals common gene alteration patterns in cancers. Theranostics. 2017;7(11):2888-2899. https://doi.org/10.7150/thno.19425. eCollection 2017 PMID: 28824723 [2] Shen QS, Han YF, Wu K, He YM, Jiang XL, Liu PS, et al. MrgprF acts as a tumor suppressor in cutaneous melanoma by restraining PI3K/Akt signaling. Signal Transduct Target Ther. 2022;7(1):147. Epub 2022/05/04. https://doi.org/10.1038/s41392-022-00945-9 PMID: 35504869 Attachments Attachment Submitted filename: Response to Reviewers.doc* https://doi.org/10.1371/journal.pone.0302061.r002
6 Mar 2024 Decision Letter - Suzie Chen, Editor PONE-D-23-27240R1GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathwayPLOS ONE Dear Dr. Zhao, 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. Minor revision has been suggested by the reviewer based on your submitted revised manuscript. Please submit your revised manuscript by Apr 20 2024 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: https://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, Suzie Chen 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. [Note: HTML markup is below. Please do not edit.] 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 #4: (No Response) ******** 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 #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #4: 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 #4: 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 #4: No ****** 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 #4: In this manuscript by Zhao and colleagues they describe the function of GPR168 in melanoma. Their mouse data suggests that GPR168 may act as a tumor suppressor in melanoma and they have shown data suggesting this but clarification and limitations of the study need to be discussed to improve the quality of the article. Comments: Data point distribution for all graphs should be included in each figure. Showing representative western immunoblots is not enough, data needs to be normalized, graphed, and individual data points must be shown. Limitations of this study must be stated in the discussion i.e human bioinformatics data used but only mouse data from one cell-line included and no human data included. Additionally, the limitations identified by the other reviewers must be stated. The comment made by the authors,”We repeated the western blotting experiments and found that both of CDK4 and cyclin D1 protein were decreased (Fig 3F).” based on reviewer 3’s comments of the fact that cyclin D1 did not decrease this is quite concerning since it suggest reproducibility issues which is why the authors should not show just the representative western immunoblots but rather both representative western immunoblots and quantifications of the western immunoblots normalized. ****** 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 #4: Yes: Kevinn Eddy ******** [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. https://doi.org/10.1371/journal.pone.0302061.r003
Revision 2
26 Mar 2024 Author Response 1. 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 #4: No Response: We feel sorry for our poor writings, however, we do invite a friend of us who is a native English speaker from the USA to help polish our article. And we hope the revised manuscript could be acceptable for you. 2. Data point distribution for all graphs should be included in each figure. Response: Thanks for the comment. We have now included data point distribution in all graphs. 3. Showing representative western immunoblots is not enough, data needs to be normalized, graphed, and individual data points must be shown. Response: Thanks for your suggestion. We have now normalized western blot data and showed in Fig 1E, Fig 2B, Fig 3H, Fig 4D. 4. Limitations of this study must be stated in the discussion i.e human bioinformatics data used but only mouse data from one cell-line included and no human data included. Additionally, the limitations identified by the other reviewers must be stated. Response: Thanks for the comment. We have now pointed this limitation in discussion (Lines 359-360). 5. The comment made by the authors, “We repeated the western blotting experiments and found that both of CDK4 and cyclin D1 protein were decreased (Fig 3F).” based on reviewer 3’s comments of the fact that cyclin D1 did not decrease this is quite concerning since it suggest reproducibility issues which is why the authors should not show just the representative western immunoblots but rather both representative western immunoblots and quantifications of the western immunoblots normalized. Response: Thanks for your comments. We repeated three times each western blot. We have now showed all western blot results in Supporting information and quantifications of the western immunoblots normalized in Fig 1E, Fig 2B, Fig 3H, Fig 4D. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0302061.r004
28 Mar 2024 Decision Letter - Suzie Chen, Editor GPR168 functions as a tumor suppressor in mouse melanoma by restraining Akt signaling pathway PONE-D-23-27240R2 Dear Dr. Zhao, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Suzie Chen 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 #4: 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 #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #4: 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 #4: 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 #4: 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 #4: All comments comments have been addressed and article should be accepted since all comments have been addressed however minor english improvements are required. ****** 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 #4: Yes: Kevinn Eddy ******** https://doi.org/10.1371/journal.pone.0302061.r005
Formally Accepted
15 May 2024 Acceptance Letter - Suzie Chen, Editor PONE-D-23-27240R2 PLOS ONE Dear Dr. Zhao, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Suzie Chen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0302061.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .